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Andrew Almond

Publications and source records attributed to Andrew Almond.

At least 19 recordsLinked to original sources
PubMed2007

Fourier transform mass spectrometry to monitor hyaluronan-protein interactions: use of hydrogen/deuterium amide exchange.

The use of Fourier transform mass spectrometry (FTMS) to monitor noncovalent complex formation in the gas phase under native conditions between the Link module from human tumor necrosis factor stimulated gene-6 (Link_TSG6) and hyaluronan (HA) oligosaccharides is reported. In particular, a titration experiment with increasing concentrations of octasaccharide (HA(8)) to protein produced a noncovalent complex with 1:1 stoichiometry when the oligosaccharide was in molar excess. However, in the presence of a molar excess of tetrasaccharide (HA(4)) nearly all proteins and oligosaccharides were observed in their unbound charge states. These results are consistent with solution-phase properties for this interaction in which HA(8), but not HA(4), supports high affinity Link_TSG6 binding. Hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS) was also utilized to investigate the level of global deuterium incorporation, over time, for Link_TSG6 in both the absence and presence of HA(8). After dilution into quenching conditions, deuterium incorporation reached limiting asymptotic values of 37 and 26 deuterons for the free and bound protein at 240 and 480 min, respectively, indicating that the oligosaccharide interferes with amide exchange on binding. To detect sequence-specific deuterium incorporation, pepsin digestion of Link_TSG6 in both the absence and presence of HA(8) was performed. A level of deuterium incorporation of 10-30% was observed for peptides analyzed in free Link_TSG6. Interestingly, HA(8) blocked some sites of proteolysis in Link_TSG6 compared to the free protein. Molecular modeling indicated that amino acids proximal to the ligand correlated with regions of the protein that were resistant to enzymatic digestion. Of the peptides that could be analyzed by H/D-EX MS in the presence of the ligand, a 30-60% reduction in deuterium incorporation, relative to the free protein, was observed, even for those sequences not directly involved in HA binding. These results support the utility of FTMS as a method for the characterization of protein-carbohydrate interactions.

Binding Sites↗
PubMed2006

Complete assignment of hyaluronan oligosaccharides up to hexasaccharides.

The glycosaminoglycan hyaluronan is involved in a diverse range of physiological and diseases processes and comprises repeated disaccharide units of N-acetyl-d-glucosamine (GlcNAc) and d-glucuronic acid (GlcA). A molecular description of the solution conformation of HA is required to account for this biology, which is best attained using nuclear magnetic resonance (NMR). NMR studies of the polymer, however, are frustrated by resonance overlap arising from the highly degenerate structure. In contrast, end-effects in oligosaccharides can produce some chemical shift dispersion, giving the possibility that their conformational properties can be measured and extrapolated to models of the polymer. We report the complete resolution and assignment of (1)H, (13)C and (15)N nuclei in hyaluronan oligosaccharides with seven different naturally occurring terminal rings. At 900MHz, all (1)H nuclei in the hexasaccharide GlcA-beta-(1-->3)-GlcNAc-beta-(1-->4)-GlcA-beta-(1-->3)-GlcNAc-beta-(1-->4)-GlcA-beta-(1-->3)-GlcNAc-OH were uniquely resolved and the two central rings were found to be a good model for the polymer environment. These assignments now allow resolved, unambiguous structural restraints to be acquired on this oligosaccharide and extrapolated to models for the solution conformation of the polymer.

Animals↗
PubMed2006

Defined megadalton hyaluronan polymer standards.

The utility of polymer standards for the calibration of average molecular mass estimates often is limited by the polydispersity--the breadth of the size distribution--of the standard. Here monodisperse synthetic hyaluronan (or hyaluronic acid [HA]) complexes in the approximately 1- to 8-megadalton (MDa) range were prepared in two steps. First, synchronized stoichiometrically controlled in vitro reactions yielded linear narrow size distribution biotinylated HA chains. Second, streptavidin protein was added at substoichiometric levels to prepare a series of complexes with one, two, three, or four HA chains per streptavidin molecule. The dendritic-like molecules approximate the mobility of natural linear HA chains on agarose gels, making the complexes useful as defined size standards for high-molecular weight HA preparations.

Adjuvants, Immunologic↗
PubMed2006

NMR spectra of oligosaccharides at ultra-high field (900 MHz) have better resolution than expected due to favourable molecular tumbling.

Nuclear magnetic resonance (NMR) remains the most promising technique for acquiring atomic-resolution information in complex carbohydrates. Significant obstacles to the acquisition of such data are the poor chemical-shift dispersion and artifacts resultant from their degenerate chemical structures. The recent development of ultra-high-field NMR (at 900 MHz and beyond) gives new potential to overcome these problems, as we demonstrate on a hexasaccharide of the highly repetitive glycosaminoglycan hyaluronan. At 900 MHz, the expected increase in spectral dispersion due to higher resonance frequencies and reduction in strong coupling-associated distortions are observed. In addition, the fortuitous molecular tumbling rate of oligosaccharides results in longer T2-values that further significantly enhances resolution, an effect not available to proteins. Combined, the resolution enhancement can be as much as twofold relative to 600 MHz, allowing all 1H-resonances in the hexasaccharide to be unambiguously assigned using standard natural-abundance experiments. The use of ultra-high-field spectrometers is clearly advantageous and promises a new and exciting era in carbohydrate structural biology.

Carbohydrate Sequence↗
PubMed2006

Hyaluronan: the absence of amide-carboxylate hydrogen bonds and the chain conformation in aqueous solution are incompatible with stable secondary and tertiary structure models.

Contradictory descriptions for the aqueous solution conformation of the glycosaminoglycan hyaluronan (HA) exist in the literature. According to hydrodynamic and simulation data, HA molecules are stiffened by a rapidly interchanging network of transient hydrogen bonds at the local level and do not significantly associate at the global level. In marked contrast, models derived from NMR data suggest that the secondary structure involves persistent hydrogen bonds and that strong associations between chains can occur to form vast stable tertiary structures. These models require an extended 2-fold helical conformation of the HA chain and specific hydrogen bonds between amide and carboxylate groups. To test these descriptions, we have used 15N-labelled oligosaccharides and high-field NMR to measure pertinent properties of the acetamido group. The amide proton chemical shift perturbation and carboxylate group pK(a) value are inconsistent with a highly populated hydrogen bond between the amide and carboxylate groups. Amide proton temperature coefficients and chemical exchange rates confirm this conclusion. Comparison of oligomer properties with polymeric HA indicates that there is no discernible difference in amide proton environment between the centre of octasaccharides and the polymer, inconsistent with the formation of tertiary structures. A [1H-1H-15N] NOESY-HSQC (heteronuclear single-quantum correlation) spectrum recorded on an HA octasaccharide revealed that amide groups in the centre are in a trans orientation and that the average solution conformation is not an extended 2-fold helix. Therefore the two key aspects of the secondary and tertiary structure models are unlikely to be correct. Rather, these new NMR data agree with descriptions from hydrodynamic and simulations data.

Amides↗
PubMed2006

The importance of including local correlation times in the calculation of inter-proton distances from NMR measurements: ignoring local correlation times leads to significant errors in the conformational analysis of the Glc alpha1-2Glc alpha linkage by NMR spectroscopy.

The experimental determination of oligosaccharide conformations has traditionally used cross-linkage 1H-1H NOE/ROEs. As relatively few NOEs are observed, to provide sufficient conformational constraints this method relies on: accurate quantification of NOE intensities (positive constraints); analysis of absent NOEs (negative constraints); and hence calculation of inter-proton distances using the two-spin approximation. We have compared the results obtained by using 1H 2D NOESY, ROESY and T-ROESY experiments at 500 and 700 MHz to determine the conformation of the terminal Glc alpha1-2Glc alpha linkage in a dodecasaccharide and a related tetrasaccharide. For the tetrasaccharide, the NOESY and ROESY spectra produced the same qualitative pattern of linkage cross-peaks but the quantitative pattern, the relative peak intensities, was different. For the dodecasaccharide, the NOESY and ROESY spectra at 500 MHz produced a different qualitative pattern of linkage cross-peaks, with fewer peaks in the NOESY spectrum. At 700 MHz, the NOESY and ROESY spectra of the dodecasaccharide produced the same qualitative pattern of peaks, but again the relative peak intensities were different. These differences are due to very significant differences in the local correlation times for different proton pairs across this glycosidic linkage. The local correlation time for each proton pair was measured using the ratio of the NOESY and T-ROESY cross-relaxation rates, leaving the NOESY and ROESY as independent data sets for calculating the inter-proton distances. The inter-proton distances calculated including the effects of differences in local correlation times give much more consistent results.

Acetylglucosamine↗
PubMed2006

Enzymatic and chemical methods for the generation of pure hyaluronan oligosaccharides with both odd and even numbers of monosaccharide units.

Hyaluronan oligosaccharides display physiological activities not associated with the polymer and are widely used to characterize hyaluronan-binding proteins. They can also be used as biocompatible starting blocks for chemical derivatization. Here we present methods for generating milligram quantities of unusual odd- and even-numbered oligosaccharides, greatly increasing the diversity of reagents for use in such studies. These methods are based upon protocols from the 1960s, at which time it was very difficult to assess the stereochemical purity of the products. To address this, products were analyzed with modern high-field nuclear magnetic resonance spectroscopy. Alkaline beta-elimination conditions previously used to remove reducing-terminal N-acetylglucosamine residues in fact introduce a significant ( approximately 30%) level of stereoisomerism in the products by alkali-catalyzed keto-enol tautomerizations. Milder alkaline conditions were used to overcome this problem, reducing the contamination to <5%. The elimination by-products from this reaction were isolated and characterized, allowing the mechanism of alkaline degradation of hyaluronan to be investigated for the first time. beta-Glucuronidase was used to remove nonreducing-terminal glucuronic acid residues from oligosaccharides. Odd-numbered oligosaccharides with terminal glucuronic acid residues isolated from hyaluronidase digests are shown to originate from acid-catalyzed acetal hydrolysis during boiling denaturation and also have significant levels of stereochemical impurities.

Alkalies↗
PubMed2006

Hyaluronan: the local solution conformation determined by NMR and computer modeling is close to a contracted left-handed 4-fold helix.

The polysaccharide hyaluronan (HA) is a ubiquitous component of the vertebrate extracellular matrix with diverse physiological roles from space-filling to acting as a scaffold for other macromolecules. The molecular interactions responsible for these solution properties have been the subject of much debate and, primarily due to the lack of residue-specific experimental data, no consensus model for the three-dimensional conformation nor dynamics of HA in solution has emerged. Here, the solution conformation of HA is investigated using molecular dynamics (MD) simulations and high-field nuclear magnetic resonance (NMR). In contrast to previous studies, MD simulations incorporated explicit water molecules and sodium ions, while NMR experiments utilized (15)N-enriched oligosaccharides to allow residue-specific information to be obtained. The resultant average conformation is predicted to be almost a contracted left-handed 4-fold helix; i.e. similar to that observed for sodium hyaluronate fibers by X-ray diffraction, but with the acetamido side-chain trans to H(2). The glycosidic linkages and acetamido side-chains are predicted to have standard deviation rotations of 13 degrees and 18 degrees around their mean conformations in free solution, respectively, and are not observed to be stabilized by strong intramolecular hydrogen bonds as X-ray fiber diffraction refinements describe for the solid-state. Rather, weak and transient hydrogen bonds that are in rapid interchange with solvent molecules are predicted. These predictions are quantitatively consistent with demanding residue-specific NMR data and correspond to an HA molecule that is rod-like as an oligosaccharide and behaves as a stiffened random coil at large molecular mass, in close agreement with previous hydrodynamic observations. This new description of the solution conformation of HA is consistent with all available experimental data and accounts for its viscoelastic space-filling properties. This representation can be used as a basis for modeling the association between HA and proteins, which will elucidate important aspects of extracellular matrix assembly.

Animals↗
PubMed2005

Towards understanding the interaction between oligosaccharides and water molecules.

Complex carbohydrates are implicated in many important biological processes, and have a strong interaction with water. This close interplay with molecular water through multiple hydroxyls may be an integral part of their emergent structure and dynamics, as selected during evolution. Using molecular dynamics simulations with explicit water the interactions at the linkages within a variety of oligosaccharides are investigated and contrasted, in order to establish correlations between linkage orientation, sugar epimerization, and water interaction. In particular, interactions at alpha linkages, and between mannose and glucose residues, that are common in oligosaccharides are considered. Sugars joined by alpha linkages at the 2-, 3-, and 6-position were found to interact via a combination of weak hydrogen-bonds and water-bridges, which is dependent on the epimerization state of the sugars. Due to their three-dimensional structure, they are also likely to interact with noncontiguous sugar residues in an oligosaccharide, which can lead to ordered structures through the exclusion of water. On the other hand, beta linkages (to 3- and 4-position) maintain strong hydrogen-bonds, have a limited ability to be involved in water-bridges, and predominantly interact with the directly attached sugars. Therefore, sequences of alpha-linked sugars form compact, branched structures that have conformational flexibility, and beta linkages form extended, relatively rigid structures, suitable for structural molecules, and at the termini of protein bound oligosaccharides. These results provide further tentative ties between chemical structure, water interactions, and the emergent form and function of specific sugars and linkages in oligosaccharides.

Carbohydrate Conformation↗
PubMed2005

Towards a structure for a TSG-6.hyaluronan complex by modeling and NMR spectroscopy: insights into other members of the link module superfamily.

The Link module from human TSG-6, a hyaladherin with roles in ovulation and inflammation, has a hyaluronan (HA)-binding groove containing two adjacent tyrosine residues that are likely to form CH-pi stacking interactions with sequential rings in the sugar. We have used this observation to construct a model of a protein.HA complex, which was then tested against existing experimental information and by acquisition of new NMR data sets of [(13)C, (15)N]HA (8-mer) complexed with unlabeled protein. A major finding of this analysis was that acetamido side chains of two GlcNAc rings fit into hydrophobic pockets on either side of the adjacent tyrosines, providing a selectivity mechanism of HA over other polysaccharides. Furthermore, two basic residues have a separation that matches that of glucuronic acids in the sugar, consistent with the formation of salt bridges; NMR experiments at a range of pH values identified protein groups that titrate due to their proximity to a free carboxylate in HA. Sequence alignment and construction of homology models for all human Link modules in their HA-bound states revealed that many of these features are conserved across the superfamily, thus allowing the prediction of functionally important residues. In the case of cartilage link protein, its two Link modules were docked together (using bound HA as a guide), identifying hydrophobic residues likely to form an intra-Link module interface as well as amino acids that could be involved in supporting intermolecular interactions between link proteins and chondroitin sulfate proteoglycans. Here, we propose a mechanism for ternary complex formation that generates higher order helical structures, as may exist in cartilage aggregates.

Acetylglucosamine↗
PubMed2005

Dynamics of hyaluronan oligosaccharides revealed by 15N relaxation.

Complex carbohydrates are considered to be flexible biomolecules, yet few experimental techniques are available to characterize their dynamics. In this study, we investigate the potential of 15N relaxation to probe the dynamics of hyaluronan oligosaccharides by adapting approaches previously applied to proteins. Unlike the 13C nucleus, 15N provides considerably enhanced spectral resolution, allowing position-specific information to be measured even in the middle of oligomers as large as decasaccharides. Moreover, isotopic incorporation maintains the 1H-15N group as an isolated spin-pair, allowing relaxation experiments to be performed and interpreted at low concentrations. A methodology is described for calculating the Lipari and Szabo model-free parameters at specific positions in hyaluronan oligomers and is used to produce a dynamic representation for the hexasaccharide. In this model, the glycosidic linkages and acetamido rotamer were determined to deviate by 18 degrees and 24 degrees from their mean positions, respectively. This approach allows the dynamic structural characterization of hyaluronan and other nitrogen-containing carbohydrates. The resultant models provide crucial insights into the physical properties and biology of these flexible molecules, which are at present poorly understood.

Carbohydrate Conformation↗
PubMed2005

Exploiting the carboxylate chemical shift to resolve degenerate resonances in spectra of 13C-labelled glycosaminoglycans.

Glycosaminoglycans (GAG) are important vertebrate extracellular matrix polysaccharides that comprise repeated units of an acidic and an N-acetylated sugar. The constituent acidic sugars are central to their biological functions, but have been largely inaccessible to NMR because the (1)H resonances overlap with those from other residues. Here, pulse sequences that address this failure are developed using (13)C-enriched oligosaccharides of the glycosaminoglycan, hyaluronan, as model systems. Two pulse sequences are presented that exploit the unique chemical shifts and scalar couplings present at the carboxylate moiety to filter out coherences from the N-acetylated sugars and produce simple spectra containing only resonances from the acidic sugars. The first sequence uses one-bond couplings to correlate the carboxylate carbon with the adjacent carbon and its directly attached proton, while the second sequence exploits a long-range coupling to correlate the carboxylate carbon with the anomeric proton and carbon of the same residue. In addition, inclusion of an isotropic mixing block into these sequences allows resonances from the otherwise degenerate ring protons to be resolved. Spectra from the hyaluronan tetra- and hexasaccharides show that all glucuronic acid (GlcA) residues can be resolved from one another, allowing nuclei to be assigned in a sequence-specific manner. However, in some spectra, resonances are observed at positions not predicted by spin-operator analysis, and simulations reveal that these additional magnetisation transfers result from strong-coupling. These experiments represent a foundation from which new structural and biochemical information can be obtained in a sequence-specific manner for the acidic sugar residues in hyaluronan and other glycosaminoglycans.

Carbohydrate Conformation↗
PubMed2004

Expression and purification of functionally active hyaluronan-binding domains from human cartilage link protein, aggrecan and versican: formation of ternary complexes with defined hyaluronan oligosaccharides.

The chondroitin sulfate proteoglycan aggrecan forms link protein-stabilized complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other chondroitin sulfate proteoglycans (i.e. versican, brevican, and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this study, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells. The recombinant human proteins were found to have properties similar to those described for the native molecules (e.g. cLP was able to form oligomers, and HA decasaccharides were the minimum size that could compete effectively for their binding to polymeric HA). Gel filtration and protein cross-linking/matrix-assisted laser desorption ionization time-of-flight peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other in solution yet formed ternary complexes with HA24. N-linked glycosylation of AG1 and VG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Surprisingly, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences in the conformation of HA stabilized on binding these proteins.

Aggrecans↗
PubMed2004

Preparation and application of biologically active fluorescent hyaluronan oligosaccharides.

We report the production of biologically active hyaluronan (HA) oligosaccharides labeled with the fluorophore 2-aminobenzoic acid (2AA). Oligosaccharides from 4 to 40 residues in length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterization of HA oligosaccharides is facilitated by 2AA derivatization because it enhances signals in MALDI-TOF MS and improves FACE (fluorophore-assisted carbohydrate electrophoresis) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC)-labeled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatized oligosaccharides, as demonstrated by their ability to compete for polymeric HA binding to the G1-domain of human recombinant versican (VG1). An electrophoretic mobility shift assay was used to study VG1 binding to labeled HA 8-, 10-, 20-, 30-, and 40-mers, and although no stable VG1 binding was observed to labeled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA(10) was estimated from densitometry analysis of the free oligosaccharide. Interactions involving HA 20-, 30-, and 40-mers (proposed to be multivalent) could also be studied using this protocol. Oligosaccharides labeled with 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

Animals↗
PubMed2004

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

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 13N 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.

Carbohydrate Conformation↗
PubMed2004

Oligosaccharides implicated in recognition are predicted to have relatively ordered structures.

Fucosylated O- and N-linked glycans are essential recognition molecules in plants and animals. To understand how they impart their functions, through interactions with proteins, requires a detailed analysis of structure and dynamics, but this is presently lacking. In this study, the three-dimensional structure and dynamics of three fucosylated oligosaccharides are investigated using a combination of high field (800 MHz) nuclear magnetic resonance and long (50 ns) molecular dynamics simulations in explicit water. Predictions from dynamics simulations were in agreement with nuclear Overhauser cross-peak intensities. Similarly, a theory of weak alignment in neutral media resulted in reasonable predictions of residual dipolar couplings for the trisaccharide fucosyllactose. However, for larger penta- and hexasaccharides (LNF-1 and LND-1), the anisotropic component of the alignment was underestimated, attributed to shape irregularities of the fucosyl branches on an otherwise linear core, being more pronounced in a singly branched than a doubly branched oligosaccharide. Simulations, confirmed by experiment, predicted fucosylated molecules that are restricted to librations about a single average conformation. This restriction is partly due to microscopic water interactions, which act to stabilize intramolecular hydrogen bonds and maintain tight and ordered conformations; a view not forthcoming from simpler, nonaqueous simulations. Such a conclusion is crucial for understanding how these molecules interact with proteins and impart their recognition properties.

Carbohydrate Conformation↗
PubMed2003

The link module from ovulation- and inflammation-associated protein TSG-6 changes conformation on hyaluronan binding.

The solution structure of the Link module from human TSG-6, a hyaladherin with important roles in inflammation and ovulation, has been determined in both its free and hyaluronan-bound conformations. This reveals a well defined hyaluronan-binding groove on one face of the Link module that is closed in the absence of ligand. The groove is lined with amino acids that have been implicated in mediating the interaction with hyaluronan, including two tyrosine residues that appear to form essential intermolecular hydrogen bonds and two basic residues capable of supporting ionic interactions. This is the first structure of a non-enzymic hyaladherin in its active state, and identifies a ligand-induced conformational change that is likely to be conserved across the Link module superfamily. NMR and isothermal titration calorimetry experiments with defined oligosaccharides have allowed us to infer the minimum length of hyaluronan that can be accommodated within the binding site and its polarity in the groove; these data have been used to generate a model of the complex formed between the Link module and a hyaluronan octasaccharide.

Amino Acid Sequence↗