Use of 15N-NMR to resolve molecular details in isotopically-enriched carbohydrates: sequence-specific observations in hyaluronan oligomers up to decasaccharides

Charles D. Blundell2, Paul L. DeAngelis3, Anthony J. Day4 and Andrew Almond1,2

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


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The glycosaminoglycan hyaluronan is a vital structural component of extracellular matrices with diverse biological functions, a molecular understanding of which requires a detailed description of secondary and tertiary solution structures. Various models of these structures have been proposed on the basis of 1H and 13C natural-abundance nuclear magnetic resonance (NMR) experiments, but resonance overlap limits further progress with these techniques. We have therefore produced 15N- and 13C- isotopically-labeled hyaluronan oligosaccharides and applied triple-resonance and 3D experiments to overcome this restriction. Spectra recorded on oligosaccharides (of lengths 4, 6, 8, 10, and 12 sugar rings), reveal that the 15N nucleus allows resolution of the amide groups in a decamer at high magnetic field, whereas 13C natural-abundance NMR can only resolve internal groups up to hexamers. Complete 15N sequence- specific assignments of these oligosaccharides indicate that the chemical shift dispersion can be explained by end-effects, which are seen even in the middle of octamers. Triple- resonance and 15N-edited 3D experiments, among the first of their kind in oligosaccharides, have been used to achieve resolution of ring 1H and 13C nuclei where not possible previously. The subtle chemical shift perturbations resolved suggest that different conformations and dynamics occur at the ends, which may contribute to the range of biological activities displayed by varying lengths of hyaluronan. 15N-NMR in carbohydrates has not received much attention before, however, this study demonstrates it has clear advantages for achieving resolution and assessing dynamic motion. These conclusions are likely to be applicable to the study of the structure and dynamics of other nitrogen-containing carbohydrates.

Key words: end-effect/15N isotope incorporation / hyaluronan / NMR / triple-resonance


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Hyaluronan (HA) is a linear, high-molecular-mass (105–107 Da) glycosaminoglycan made from a repeated disaccharide unit of glucuronic acid (GlcA) and N-acetyl-glucosamine (GlcNAc) (Figure 1). It is found in the extracellular matrix of all adult vertebrate tissues and has diverse biological roles (Fraser et al., 1997Go;Tammi et al., 2002Go). These include acting as a vital structural component of connective tissues (Morgelin et al., 1988Go), the formation of loose hydrated matrices that allow cellular migration and division (Toole 2001Go, 2002Go), immune cell adhesion and activation (Gal et al., 2003Go; Kim et al., 2000Go), and a role in intracellular signaling (Turley et al., 2002Go). Furthermore, increases in the concentration of HA have been shown to be important in such processes as ovulation (Fulop et al., 1997Go; Salustri et al., 1995Go), development (Camenisch and McDonald, 2000Go; Toole 1997Go), and several diseases, such as inflammatory bowel disease (de La Motte et al., 1999Go) and various cancers (Wang et al., 1996Go). It is believed that many of these functions are mediated by HA-binding proteins (Day and Prestwich, 2002Go), but free HA is no less important, providing filtering, space-filling, and lubricating functions (e.g., in synovial fluid) (Fraser et al., 1997Go). In addition, low- molecular-weight HA and its oligosaccharides have activities not associated with the parent molecule, such as inducing expression of inflammatory mediators in alveolar macrophages (Horton et al., 1999Go; McKee et al., 1996Go; Noble et al., 1996Go), promoting angiogenesis (Rahmanian et al., 1997Go; West et al., 1985Go), induction of immunophenotypic maturation of dendritic cells (Termeer et al., 2000Go, 2002Go) and inhibiting tumor growth in vivo (Zeng et al., 1998Go), but the molecular basis for these differences is not understood.



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Fig. 1. The repeated disaccharide unit of hyaluronan, which is comprised of glucuronic acid (GlcA) and N-acetyl-glucosamine (GlcNAc) joined by ß(1 -> 3) and ß(1 -> 4) linkages. The nomenclature for identifying the carbon atoms is indicated.

 
High-molecular-weight HA is extremely soluble, generating solutions with a highly viscoelastic nature at low concentrations. Hydrodynamic measurements have concluded that polymeric HA acts as a stiffened, wormlike coil, with a persistence length of between 4 and 6 nm (Cleland, 1977Go; Laurent et al., 1960Go). HA chains never form true (i.e., cuttable) gels at physiological pH even when the concentration or chain length are increased. Rather, they form increasingly viscoelastic solutions that always flow, indicating that the chains do not form strong, stable intermolecular associations (Almond et al., 1998aGo; Day and Sheehan, 2001Go). Intramolecular hydrogen bonds between successive sugar moieties have been proposed to be responsible for the fibrous structure of the polymer and its physicochemical behavior in aqueous solution (Almond et al., 1997Go; Heatley and Scott, 1988Go; Mikelsaar and Scott, 1994Go; Scott et al., 1981Go). Although evidence has been put forward that hydrogen bonds across the glycosidic linkage stiffen the HA molecule (Atkins and Sheehan, 1972Go; Scott and Heatley, 2002Go; Scott and Tigwell, 1978Go), analysis of these hydrogen bonds in HA oligosaccharides in water using molecular dynamics simulations (Almond et al., 1997Go, 1998bGo) suggest that they are less stable than has been previously proposed because it appears that intramolecular and water hydrogen-bonding partners frequently interchange on the picosecond time scale.

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., 1996Go) and 1H nuclei in the tetramer (HA4) (Livant et al., 1992Go; Toffanin et al., 1993Go) have been performed, although not without some discrepancies. Additional unique carbon nuclei assignments have been made for the ends of octasaccharides (Cowman et al., 1996Go; Holmbeck et al., 1994Go), and the dynamic motion at various positions along the chain assessed, demonstrating that end-effects are significant in HA6 (Cowman et al., 2001Go). 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., 1994Go). 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, 1999Go, 2002Go). 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., 1981Go) but refuted by studies in water (Cowman et al., 1984Go; Sicinska et al., 1993Go). 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., 1994Go), or some conformations have been singled out as a result of the particular modeling technique employed (Almond et al., 1998; Mikelsaar and Scott, 1994Go), 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, 1998Go). 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., 2000Go). 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.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Purification of unlabeled, 15N- and 15N-, 13C-labeled HA oligosaccharides
High-molecular-weight unlabeled and isotopically labeled HA were digested using ovine testicular hyaluronidase, resulting in the series of even-numbered oligosaccharides containing GlcNAc at the reducing terminus (see Mahoney et al., 2001Go). Oligomers from the tetrasaccharide (HA4) up to the dodecamer (HA12) were separated with anion-exchange chromatography (Figure 2), desalted, and lyophilized, yielding the ammonium salts (see Materials and methods). From 100 mg of digested HA, ~90 mg of products were typically recovered, distributed as 35 mg HA4, 25 mg HA6, 15 mg HA8, 10 mg HA10, and 5 mg HA12.



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Fig. 2. Anion-exchange HPLC of HA digest with testicular hyaluronidase (unlabeled material). Oligosaccharides of increasing length are eluted, beginning with the tetrasaccharide (HA4); the initial peak corresponds to acetate (Ac) from the digestion buffer. At the end of the NaCl gradient, the step to high NaCl concentration results in the elution of HA14 and all higher-molecular-mass products. The conductivity (indicating NaCl concentration) is shown with a dashed line.

 
Both NMR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify the oligosaccharide preparations. Negative-ion mass spectra were collected for both unlabeled and isotopically-labeled samples and the masses found to be in excellent agreement with those expected (Table I). The increased masses of the labelled oligomers are clearly visible (see Figure 3A), and correspond to ~100% incorporation of both 15N and 13C. Comparison of the intensities of the {alpha}, ß, and internal amide resonances in 1D NMR spectra (as Cowman et al., 1984Go) allowed the identifications made by MALDI-TOF to be confirmed. High-resolution 1H,15N–heteronuclear single quantum coherence (HSQC) spectra were also found to be distinctive for each length of oligosaccharide up to HA10 (discussed below).


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Table I. Observed and theoretical molecular masses of unlabeled and isotopically labeled HA oligosaccharides

 


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Fig. 3. (A) MALDI-TOF spectra of unlabeled (bottom) and 15N-labeled HA6 (top), showing the monoisotopic mass of the monovalent anions. Because each disaccharide in HA6 contains one nitrogen atom, 15N-incorporation results in a 3-Da increase, as observed. Peaks i and ii correspond to the mono- and disodium adducts, respectively. (B) Fluorescent polyacrylamide gel electrophoresis of purified oligosaccharides from HA4 to HA10 compared against a sample from the unpurified digest after 120 min reaction time.

 
Analysis of oligosaccharide purity
Purified HA oligomer preparations were run on an analytical FPLC Mono-Q HR5/5 column to determine the extent of contamination with other lengths of HA. As can be seen from Figure 4, each length of oligosaccharide was separated from the others to >95% purity. This was further confirmed by polyacrylamide gel electrophoresis of fluorescently labeled oligomers (Figure 3B) and the MALDI-TOF analyses (Figure 3A). The presence of non-UV-absorbing small molecules in the preparations was assessed using both 1D and 2D NMR spectra and found to be negligible. The fraction by mass of bound H2O in our lyophilized HA preparations was measured by reconstituting material in 99.9 atom % D2O and comparing the intensity of the residual H2O NMR peak against a standard curve of known H2O %. It was found that H2O molecules retained in lyophilized pellets comprise <2% of their mass, irrespective of the oligosaccharide length.



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Fig. 4. Analysis of purified HA oligosaccharide preparations by anion-exchange HPLC. A sample from the unpurified digest (unlabeled material, after 120 min reaction) containing the full range of oligomer lengths (top), is compared against runs of each purified oligosaccharide from HA4 to HA10 (lower panels).

 
Sequence-specific assignment of internal HN groups
Using these highly characterized oligosaccharide preparations, NMR samples were generated for HA4, HA6, HA8, and HA10, both with and without 15N label. 1H,15N-HSQC and 1D spectra recorded on these samples at 750 MHz are shown in Figure 5A. HA oligosaccharides contain one amide proton per disaccharide unit (in the GlcNAc ring, refer to Figure 1), which could therefore be expected to give one distinct resonance each, with the caveat that the reducing terminal GlcNAc ring produces two resonances (one each for {alpha}- and ß-anomers). However, as shown by the 1D spectra, whereas the {alpha}- and ß-anomers are resolved by virtue of their different chemical shifts (arising from their distinct chemical bonding), the internal amide protons (which have identical local chemical bonding) have virtually indistinguishable chemical shifts. In fact, in 1D spectra the two internal groups in HA6 are barely resolved at 750 MHz. Employment of 15N-labeling however, allows distinct resonances to be resolved for every HN group within HA oligomers up to HA8, and for several groups within HA10 (Figure 5B). The 3JHNH2 coupling constant was measured for each resonance in these spectra and found to be 9.5 ± 0.5 Hz at each position, as assumed previously (see Almond et al., 1998bGo).



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Fig. 5. 1H,15N-HSQC spectra of HA oligosaccharides of different lengths. (A) Complete spectra of HA4, HA6, HA8, and HA10, with assignments indicated (refer to Figure 6 for nomenclature). The corresponding 1D spectrum is shown for each length of HA above the HSQC, revealing the resolution gained by employing the 15N-dimension. (B) Overlay of a portion of the spectra, showing the relationship between the internal HN resonances in different oligosaccharides (coloured as in A).

 


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Fig. 6. Nomenclature of GlcNAc rings in HA oligosaccharides of differing length. Rings are designated using the greek alphabet from both termini in towards the middle; the reducing terminus, indicated with -OH, begins with {alpha}/ß (corresponding to {alpha}- and ß-anomers) and proceeds through the alphabet on successive GlcNAc rings (gray), whereas the nonreducing terminus begins with {omega} and regresses. GlcA rings are shown in white. The principal advantage of this system is that rings in the same environment in different lengths of HA take the same designation.

 
By comparing peaks across the series of oligosaccharides, it is possible to uniquely assign each amide resonance to a specific ring in each oligosaccharide. The nomenclature devised to refer to the amide groups at each position in an oligosaccharide is shown in Figure 6. The {alpha}- and ß-anomers are readily identified in each oligomer by their reduced intensities relative to the internal amides and 1H chemical shifts, which have been reported before (Cowman et al., 1984Go). In HA4, the remaining resonance clearly arises from the internal amide group ({omega}HN). Neither internal resonance in HA6 directly overlaps with that of {omega}HN in HA4 (Figure 5B), however the downfield one (15N) is the obvious candidate for {omega}HN in HA6 because it is considerably closer. The remaining upfield (15N) resonance in HA6 is therefore assigned to the {gamma} ring. Peaks in HA8 directly overlay with the {omega}HN and {gamma}HN resonances in HA6 and are assigned accordingly; the new resonance is therefore that of {psi}HN. HA10 has a very similar spectrum to HA8, although the {psi}HN peak is now overlapping with a new peak (assigned to {delta}HN) that only slightly differs in 15N and 1H chemical shifts (~10 ppb and ~5 ppb, resolvable with very strong window functions). A 1H,15N-HSQC spectrum recorded on HA12 was found to be identical to that of HA10 (supplementary Figure 1) except that the peak intensity at {delta}HN was seen to be relatively higher, indicating that the next internal group ({chi}HN) is in an indistinguishable environment at this field strength.

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 {omega} ring across the series), but they are too small to be measured precisely from these experiments.


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Table II. 1H, 15N chemical shift assignments (ppm) for HA oligosaccharides of different lengths

 
Use of the 15N-label to specifically assign nuclei in internal GlcNAc rings of HA6
Total resolution of the amide groups in the 1H,15N-HSQC has also allowed the use of 3D spectroscopy to resolve nuclei within specific GlcNAc rings that otherwise severely overlap in 2D spectra. From the 1H,15N–total correlation spectroscopy (TOCSY)–HSQC spectrum of HA6 (600 MHz) (Figure 7), it is clear that the H1, H2, and H3 protons of the internal GlcNAc rings ({gamma}, {omega}) have slightly different chemical shifts, which cannot be resolved even in a 2D TOCSY spectrum recorded at 750 MHz. The largest variation in chemical shift is seen for the H4 protons, which differ by ~10 ppb between the {omega} and {gamma} rings.



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Fig. 7. Employment of 15N-filtering in 3D spectra allows unprecedented resolution of hydrogen nuclei within HA6. Strips from a 2D TOCSY (750 MHz) are compared against those from a 1H,15N-TOCSY-HSQC (600 MHz). The amide 1H and 15N chemical shifts are indicated for each ring; proton resonances are labelled as Figure 1, following the assignments of Toffanin et al. (1993)Go.

 
A 15N-, 13C-labeled sample of HA6 was also prepared in this study, which permitted the further investigation of its GlcNAc side chain environments by triple-resonance NMR. Using HNCO and HNCA spectra, it was possible to uniquely resolve the C2, C7, and C8 atoms associated with each internal amide group (Figure 8), whereas a 1H,15N–NOE spectroscopy (NOESY)–HSQC spectrum was used to measure the H8 (methyl) chemical shift; the assignments of all nuclei in the GlcNAc side chains of HA6 are given in Table III. No detectable differences in chemical shift were seen between the methyl groups (C8, H8) or carbonyl carbons (C7) of the internal GlcNAc rings. However, the C2 chemical shifts of these rings are slightly different (~70 ppb), consistent with the slight variation in chemical shift seen for the corresponding H2 protons in the 1H,15N-TOCSY-HSQC spectrum.



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Fig. 8. Strip plot of HNCO (top) and HNCA (bottom) spectra of HA6, correlating the amide groups (indicated below strips) to the C7, C2, and C8 carbons (nomenclature as in Figure 1). The C2 resonances in the HNCA spectrum are split into triplets by the 1JCC couplings to C1 and C3 (each ~41 Hz).

 

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Table III. Assignments of 13C and 1H nuclei within the N-acetyl side chains of HA6

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The observation that 15N chemical shifts tend to a minimum at {delta}HN (Table II, Figure 5) indicates that this value is the typical chemical shift found in the polymer. Furthermore, because the local chemical bonding of the {psi}HN, {gamma}HN, and {omega}HN groups are no different from that of {delta}HN, the chemical shift differences between each of these groups and the minimum probably arise from end-effects of some kind. Therefore, because the {delta}HN chemical shift is first observed in HA10, it is apparent that end-effects are experienced even in the middle of oligomers as long as HA8 and should be considered to be significant in HA6 and HA4.

The chemical shift difference seen between {delta}HN and {omega}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 {delta}HN and {psi}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 {omega}HN group, which is adjacent to the terminal ring). The chemical shift difference observed at {gamma}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 {omega}HN position, which at first sight seems to be anomalous, can now be seen to be the typical {omega}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 {gamma}N peaks are split into distinct populations from the {alpha}- 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 {gamma}HN and {omega}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., 1998bGo). In this context, the difference observed at the H4 proton between {gamma} and {omega} 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., 1997Go, 1998bGo, 2000Go).

Chain mobility at specific positions within HA oligosaccharides has been investigated by Cowman et al. (2001)Go, 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., 1998bGo, 2000Go). 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, 1994Go) 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 {gamma} and {omega} 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., 1993Go) and HA6 (13C: Cowman et al., 1996Go) and not without some remaining disagreement (Livant et al., 1992Go; Toffanin et al., 1993Go). 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 protein–HA 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., 2004Go; Ghatak et al., 2002Go; Sugahara et al., 2003Go; Termeer et al., 2000Go). 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., 2000Go), although the structure indicates that the binding site could only accommodate five or six rings (Blundell et al., 2003Go); 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., 2003Go).

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.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Digestion
For the preparation of unlabeled oligosaccharides, 1 g of medical-grade HA (Genzyme Hylumed Medical, Cambridge, MA, Mr 0.5–1.5 M Da) was dissolved in 100 ml digest buffer (150 mM NaCl, 100 mM NaAc, adjusted to pH 5.2 with glacial acetic acid) and incubated at 37°C. Ovine testicular hyaluronidase (Calbiochem, Darmstadt, Germany) was added (100 kU) and the reaction stopped after 180 min by boiling for 10 min. A 1.5-ml sample was taken at 120 min; 1.5 ml was also allowed to react for 24 h, taking the reaction to exhaustion. Precipitated protein was removed by centrifugation and filtration.

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., 2003Go; DeAngelis et al., 1998Go), 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., 2002Go). 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 mM–100 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 mM–100 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)Go. 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 18–39 ([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 {alpha} 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., 1995Go) 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., 1995Go).


    Acknowledgements
 
We would like to thank Prof. Iain Campbell for critical review of the manuscript and Dr. Jonathan Boyd for many stimulating discussions. This work was funded by a Biotechnology and Biological Sciences Research Council Sir David Phillips research fellowship. A.J.D. acknowledges the support of the Medical Research Council and P.L.D. the National Science Foundation MCB-9876193.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: andrew.almond{at}bioch.ox.ac.uk


    Abbreviations
 
ACN, acetonitrile; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; HA, hyaluronan; HSQC, heteronuclear single quantum coherence; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TFA, trifluoroacetic acid; TOCSY, total correlation spectroscopy


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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