Search

The carbohydrate hyaluronan (or hyaluronic acid, HA) is found in all human tissues and biofluids where it has wide-ranging functions in health and disease that are dictated by both its abundance and size. Consequently, hyaluronan evaluation in physiological samples has significant translational potential. Although the analytical tools and techniques for probing other biomolecules such as proteins and nucleic acids have become standard approaches in biochemistry, those available for investigating hyaluronan are less well established. In this review, we survey methods related to the assessment of native hyaluronan in biological specimens, including protocols for separating it from biological matrices and technologies for determining its concentration and molecular weight.

Hyaluronan (HA) is an essential carbohydrate in vertebrates that is a potentially robust bioindicator due to its critical roles in diverse physiological functions in health and disease. The intricate size-dependent function that exists for HA and its low abundance in most biological fluids have highlighted the need for sensitive technologies to provide accurate and quantitative assessments of polysaccharide molecular weight and concentration. We have demonstrated that solid state (SS-) nanopore technology can be exploited for this purpose, given its molecular sensitivity and analytical capacity, but there remains a need to further understand the impacts of experimental variables on the SS-nanopore signal for optimal interpretation of results. Here, we use model quasi-monodisperse HA polymers to determine the dependence of HA signal characteristics on a range of SS-nanopore measurement conditions, including applied voltage, pore diameter, and ionic buffer asymmetry. Our results identify important factors for improving the signal-to-noise ratio, resolution, and sensitivity of HA analysis with SS-nanopores.

BackgroundTNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties.MethodsHA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3),TSG-6, and hyaluronan-degrading enzyme (HYAL2,HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively.ResultsTNF-α concentrations were greater in OA synovial fluid, andTSG6expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity.ConclusionsSynovial fluid TNF-α concentrations, synovial membrane and cartilageTSG6gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA.

A solid phase adsorption method for selective isolation of hyaluronan (HA) from biological samples is presented. Following enzymatic degradation of protein, HA can be separated from sulfated glycosaminoglycans, other unsulfated glycosaminoglycans, nucleic acids, and proteolytic fragments by adsorption to amorphous silica at specific salt concentrations. The adsorbed HA can be released from silica using neutral and basic aqueous solutions. HA ranging in size from ∼9 kDa to MDa polymers has been purified by this method from human serum and conditioned medium of cultured cells.

Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the current state of the art., Involved in various diseases, hyaluronic acid is an important indicator of pathophysiology. Here, the authors report on a solid-state nanopore for the detection of the molecular weight and abundance of hyaluronic acid and demonstrate the system by studying an equine model of osteoarthritis

Glycosaminoglycans (GAGs) have therapeutic potential in areas ranging from angiogenesis, inflammation, hemostasis and cancer. GAG bioactivity is conferred by intrinsic structural features, such as disaccharide composition, glycosidic linkages and sulfation pattern. Unfortunately, the in vitro enzymatic synthesis of defined GAGs is quite restricted by a limited understanding of current GAG synthases and modifying enzymes. Our work provides insights into GAG-active enzymes through the creation of sulfated oligosaccharides, a new polysaccharide and chimeric polymers. We show that a C6-sulfonated uridine diphospho (UDP)-glucose (Glc) derivative, sulfoquinovose, can be used as an uronic acid donor, but not as a hexosamine donor, to cap hyaluronan (HA) chains by the HA synthase from the microbe Pasteurella multocida. However, the two heparosan (HEP) synthases from the same species, PmHS1 and PmHS2, could not employ the UDP-sulfoquinovose under similar conditions. Serendipitously, we found that PmHS2 co-polymerized Glc with glucuronic acid (GlcA), creating a novel HEP-like polymer we named hepbiuronic acid [-4-GlcAβ1-4-Glcα1-]n. In addition, we created chimeric block polymers composed of both HA and HEP segments; in these reactions GlcA-, but not N-acetylglucosamine-(GlcNAc), terminated GAG acceptors were recognized by their noncognate synthase for further extension, likely due to the common β-linkage connecting GlcA to GlcNAc in both of these GAGs. Overall, these GAG constructs provide new tools for studying biology and offer potential for future sugar-based therapeutics.

Background: The polysaccharide hyaluronan is organized through interactions with the protein TSG-6 during inflammation and ovulation. Results: NMR spectroscopy on TSG-6 in the presence of defined sugars provided restraints that allowed modeling of a refined hyaluronan/TSG-6 complex. Conclusion: TSG-6 binding causes bending of hyaluronan that explains its condensation of this polysaccharide. Significance: This provides novel structural insights into protein-hyaluronan interactions., Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was 13C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.

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 1H resonances overlap with those from other residues. Here, pulse sequences that address this failure are developed using 13C-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. Copyright © 2005 John Wiley & Sons, Ltd.

Hyaluronan (HA) is a large glycosaminoglycan produced by hyaluronan synthases (HAS), enzymes normally active at plasma membrane. While HA is delivered into the extracellular space, intracellular HA is also seen, mostly in vesicular structures, but there are also reports on its presence in the cytosol and specific locations and functions there. We probed the possibility of HA localization and functions in cytosol by microinjecting fluorescent HA binding complex (fHABC), HA fragments and hyaluronidase (HYAL) into cytosol. Microinjection of fHABC did not reveal HA-specific intracellular binding sites. Likewise, specific cytosolic binding sites for HA were not detected, as microinjected fluorescent HA composed of 4-8 monosaccharide units (HA4-HA8) were evenly distributed throughout the cells, including the nucleus, but excluded from membrane-bound organelles. The largest HA tested (∼HA120 or ∼25 kDa) did not enter the nucleus, and HA10-HA28 were progressively excluded from parts of nuclei resembling nucleoli. In contrast, HA oligosaccharides endocytosed from medium remained in vesicular compartments. The activity of HA synthesis was estimated by measuring the HA coat on green fluorescent protein (GFP)-HAS3-transfected MCF-7 cells. Microinjection of HA4 reduced coat size at 4 h, but increased at 24 h after injection, while larger HA-oligosaccharides and HYAL had no influence. As a positive control, microinjection of glucose increased coat size. In summary, no evidence for the presence or function of HA in cytosol was obtained. Also, the synthesis of HA and the active site of HAS were not accessible to competition, binding and degradation by cytosolic effectors, while synthesis responded to increased substrate supply.

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.

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 (PDB code 2BVK) 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° and 18° 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.

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.

Pasteurella multocida is a pathogenic Gram-negative bacterial species that infects a wide variety of animals and humans. A notable morphological feature of many isolates is the extracellular capsule. The ability to remove the capsule by treatment with certain glycosidases has been utilized to discern various capsular types called A, D and F. Based on this preliminary evidence, these microbes have capsules made of glycosaminoglycans, linear polysaccharides composed of repeating disaccharide units containing an amino sugar. Glycosaminoglycans are also abundant components of the vertebrate extracellular matrix. It has been shown previously that the major Type A capsular material was hyaluronan (hyaluronic acid). We report that the Type D polymer is an unmodified heparin (N-acetylheparosan) with a -->4)-beta-D-Glcp-UA-(1-->4)-alpha-D-Glcp-NAc-(1--> repeating unit and the Type F polymer is an unmodified chondroitin with a -->4)-beta-D-Glcp-UA-(1-->3)-beta-D-Galp-NAc-(1--> repeating unit. The monosaccharide compositions, disaccharide profiles, and 1H NMR analyses are consistent with these identifications. The molecular size of the Pasteurella polymers is approximately 100-300 kDa as determined by gel electrophoresis and multi-angle laser light scattering; this size is much greater than the 10-30 kDa size of the analogous polymers isolated from animal tissues. The glycosaminoglycan capsular polymers are relatively non-immunogenic virulence factors that enhance microbial pathogenicity.

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.

We previously reported that the chlorella virus PBCV-1 genome encodes an authentic, membrane-associated glycosyltransferase, hyaluronan synthase (HAS). Hyaluronan, a linear polysaccharide chain composed of alternating beta1,4-glucuronic acid and beta1, 3-N-acetylglucosamine groups, is present in vertebrates as well as a few pathogenic bacteria. Studies of infected cells show that the transcription of the PBCV-1 has gene begins within 10 min of virus infection and ends at 60-90 min postinfection. The hyaluronan polysaccharide begins to accumulate as hyaluronan-lyase sensitive, hair-like fibers on the outside of the chlorella cell wall by 15-30 min postinfection; by 240 min postinfection, the infected cells are coated with a dense fibrous network. This hyaluronan slightly reduces attachment of a second chlorella virus to the infected algae. An analysis of 41 additional chlorella viruses indicates that many, but not all, produce hyaluronan during infection.

In many cases, the cellular response to hyaluronan (HA) depends on the molecular weight (MW) of the polymer chain. Most HA preparations from Nature or its derivatives possess wide size distributions called polydisperse. New chemoenzymatic synthesis technology allows the production of very narrow size distribution polymers called monodisperse. The use of stoichiometrically controlled and synchronized polymerization reactions allows an assortment of new HA reagents in the range of 10 kDa to 2,500 kDa for answering HA biology questions or potentially treating disease.

Sequence analysis of the 330-kilobase genome of the virus PBCV-1 that infects a chlorella-like green algae revealed an open reading frame, A98R, with similarity to several hyaluronan synthases. Hyaluronan is an essential polysaccharide found in higher animals as well as in a few pathogenic bacteria. Expression of the A98R gene product in Escherichia coli indicated that the recombinant protein is an authentic hyaluronan synthase. A98R is expressed early in PBCV-1 infection and hyaluronan is produced in infected algae. These results demonstrate that a virus can encode an enzyme capable of synthesizing a carbohydrate polymer and that hyaluronan exists outside of animals and their pathogens.

Multiple glycosyltransferases (GTases) that produce glycosaminoglycans (GAGs) have been identified; -several distinct putative architectures and catalytic abilities have been noted from microbes and vertebrates. Here the preparation and use of a class of enzymes (Class II) from the gram-negative bacterium, Pasteurella multocida, with utility in chemoenzymatic synthesis of GAGs is described. The analyses of sugar products include thin layer chromatography (TLC), matrix-assisted laser desorption ionization mass spectroscopy (MALDI-ToF MS), and polyacrylamide gel electrophoresis (PAGE). The related Chapter 18 is focused on larger molecular weight GAGs analyses as well as the Class I membrane streptococcal and mammalian HA synthases.

Hyaluronic acid (HA), a linear polysaccharide composed of alternating glucuronic acid and N-acetylglucosamine residues, is an essential molecule of higher vertebrates. The fowl cholera pathogen Pasteurella multocida Carter Type A also produces HA in the form of an extracellular capsule in order to evade host defenses. HA synthase activity could be obtained from cell-free membrane preparations of P. multocida. The enzyme utilized UDP-sugar precursors of HA in the presence of Mg2+ or Mn2+ at neutral pH. Mn2+ at 1 mM stimulated approximately 2-fold more incorporation than Mg2+ at 10 mM. On the other hand, the analogous enzyme from group A Streptococcus, HasA, is stimulated more by Mg2+ than Mn2+. The apparent Michaelis constants, K(M), of the P. multocida HA synthase for UDP-N-acetylglucosamine and UDP-glucuronic acid were estimated to be approximately 75 and approximately 20 microM, respectively, in the presence of Mg2+, which suggests that the substrates are bound with 2-3-fold higher affinity than by the HasA enzyme. The rate enhancement observed with Mn2+ is apparently not due to better binding of the sugar nucleotide precursors complexed to Mn ion because the K(M) value, a measure of substrate affinity, increases by 25-50% in comparison to Mg2+. In summary, the HA synthase from P. multocida, a Gram-negative bacterium, has kinetic optima distinct from those of HasA, the analog from the Gram-positive group A Streptococcus.

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).

One of the virulence factors of group A streptococci is the hyaluronic acid polysaccharide capsule. A rapid method for ascertaining the status of the capsule phenotype in Streptococcus pyogenes is described. Bacteria with a capsule have a lower buoyant density than acapsular or hyaluronidase-treated cells. Early log phase cultures were underlaid with 65% Percoll and centrifuged at 500–1000 g for 5 min. Upon visual examination, encapsulated cells were observed at the interface, whereas acapsular cells appeared in the pellet. Cultures that produced at least 7 μg/ml of hyaluronic acid per A 600 unit of cells were detected at the interface; this level of polysaccharide is only about 0.5%–4% of that found for most mucoid strains. Therefore, this procedure can detect capsules around strains that do not appear to be encapsulated by light microscopy or do not possess mucoid colony morphology. Furthermore, this method reduces dependence on other expensive assays that use labile radioactive reagents to detect hyaluronic acid .

Hyaluronan synthases (HASs) are glycosyltransferases that catalyze polymerization of hyaluronan found in vertebrates and certain microbes. HASs transfer two distinct monosaccharides in different linkages and, in certain cases, participate in polymer transfer out of the cell. In contrast, the vast majority of glycosyltransferases form only one sugar linkage. Although our understanding of HAS biochemistry is still incomplete, very good progress has been made since the first genetic identification of a HAS in 1993. New enzymes have been discovered, and some molecular details have emerged. Important findings are the lipid dependence of Class I HASs, the function of HASs as protein monomers, and the elucidation of mechanisms of synthesis by Class II HAS. We propose three classes of HASs based on differences in protein sequences, predicted membrane topologies, potential architectures, mechanisms, and direction of polymerization.

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (approximately 10(2-4) sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.

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 pKa 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.

The hasA gene from Streptococcus equisimilis , which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis , resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis -derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.

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.

Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative virulence factor in certain pathogenic bacteria, is an extended linear polymer composed of alternating units of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). Uncertainty regarding the mechanism of HA biosynthesis has included the directionality of chain elongation, i.e. whether addition of monosaccharide units occurs at the reducing or non-reducing terminus of nascent chains. We have investigated this problem using yeast-derived recombinant HA synthases from Xenopus laevis (xlHAS1) and from Streptococcus pyogenes (spHAS). The enzymes were incubated with UDP-[3H]GlcUA and UDP-[14C]GlcNAc, under experimental conditions designed to yield HA chains with differentially labeled reducing-terminal and non-reducing terminal domains. Digestion of the products with a mixture of beta-glucuronidase and beta-N-acetylglucosaminidase exoenzymes resulted in truncation of the HA chain strictly from the non-reducing end and release of labeled monosaccharides. The change in 3H/14C ratio of the monosaccharide fraction, during the course of exoglycosidase digestion, was interpreted to indicate whether sugar units had been added at the reducing or non-reducing end. The results demonstrate that the vertebrate xlHAS1 and the bacterial spHAS extend HA in opposite directions. Chain elongation catalyzed by xlHAS1 occurs at the non-reducing end of the HA chain, whereas elongation catalyzed by spHAS occurs at the reducing end. The spHAS is the first glycosyltransferase that has been unanimously demonstrated to function at the reducing end of a growing glycosaminoglycan chain.

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.

We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (beta 4-glucuronic acid-beta 3-N-acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (approximately 10-20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (approximately 1-100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicant-tagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.

Hyaluronan (or hyaluronic acid or hyaluronate; HA) is a polysaccharide found in the extracellular matrix of vertebrate tissues and in the surface coating of certain Streptococcus and Pasteurella bacterial pathogens. At least one algal virus directs its host to produce HA on the cell surface early in infection. HA synthases (HASs) are the enzymes that polymerize HA using uridine diphospho-sugar precursors. In all known cases, HA is secreted out of the cell; therefore, HASs are normally found in the outer membranes of the organism. In the last 6 years, the HASs have been molecularly cloned from all the above sources. They were the first class of glycosyltransferases identified in which a single polypeptide species catalyzes the transfer of two different monosaccharides; this finding is in contrast to the usual ‘single enzyme, single sugar’ dogma of glycobiology. There appear to be two distinct classes of HASs based on differences in amino acid sequence, topology in the membrane, and reaction mechanism. This review discusses the current state of knowledge surrounding the molecular details of HA biosynthesis and summarizes the possible evolutionary history of the HASs.

We have characterized the hyaluronan (HA) synthase activity of the Xenopus DG42 gene product in vitro. The recombinant enzyme produced in yeast does not possess a nascent HA chain and, therefore, is an ideal model system for kinetic studies of the synthase's glycosyltransferase activity. The enzymatic rate was optimal from pH 7.6 to 8.1. Only the authentic sugar nucleotide precursors, UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc), were utilized to produce a large molecular weight polymer. UDP-glucose or the galactose epimers of the normal substrates did not substitute. The Michaelis constant, Km, of recombinant DG42 in membranes was 60 +/- 20 and 235 +/- 40 microM for UDP-GlcA and UDP-GlcNAc, respectively, which is comparable to values obtained previously from membranes derived from vertebrate cells. The apparent energy of activation for HA elongation is about 15 kilocalories/mol. DG42 polymerizes HA at average rates of about 80 to 110 monosaccharides/s in vitro. The resulting HA polysaccharide possessed molecular weights spanning 2 x 10(6)-10(7) Da, corresponding to about 10(4) sugar residues. This is the first report characterizing a defined eukaryotic enzyme that can produce a glycosaminoglycan.

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.

We demonstrate in this report that the Xenopus DG42 gene product made in the yeast Saccharomyces cerevisiae can synthesize authentic high molecular weight hyaluronan (hyaluronic acid; HA) in vitro. Saccharomyces are eukaryotes that do not naturally produce HA or any other molecules known to contain glucuronic acid. Therefore bakers' yeast is a good model system to determine the enzymatic activity of the DG42 protein, which is the topic of recent debate. Membrane extracts prepared from cells expressing DG42 encoded on a plasmid incorporated [14C]glucuronic acid and N-[3H]acetylglucosamine from exogenously supplied UDP-sugar nucleotides into a high molecular mass (10(6) to 10(7) Da) polymer in the presence of magnesium ions. Both sugar precursors were simultaneously required for elongation. Control extracts prepared from cells with the vector plasmid alone or the DG42 cDNA in the antisense orientation did not display this activity. The double-labeled polysaccharide product synthesized in vitro was deemed to be HA by enzymatic analyses; specific HA lyase could degrade the polymer, but it was unaffected by protease or chitinase treatments. The fragments generated by HA lyase were identical to fragments derived from authentic vertebrate HA as compared by high performance liquid chromatography. We conclude that DG42 is a membrane-associated HA synthase capable of transferring both glucuronic acid and N-acetylglucosamine groups.