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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.

In this age of translational science, blossoming biotechnology and increasingly elusive federal funding, a working knowledge of commercialization beyond typical academic training is necessary. Here, some issues and initial strategies to help principal investigators, postdoctoral fellows and students considering the route from discovery to invention to commercialization in the glycobiology field. Common myths are addressed, and tips and resources are provided to help start down this path.

Nanoparticle modification with poly(ethylene glycol) (PEG) is a widely used surface engineering strategy in nanomedicine. However, since the artificial PEG polymer may adversely impact nanomedicine safety and efficacy, alternative surface modifications are needed. Here, we explored the "self" polysaccharide heparosan (HEP) to prepare colloidally stable HEP-coated nanoparticles, including gold and silver nanoparticles and liposomes. We found that the HEP-coating reduced the nanoparticle protein corona formation as efficiently as PEG coatings upon serum incubation. Liquid chromatography-mass spectrometry revealed the protein corona profiles. Heparosan-coated nanoparticles exhibited up to 230-fold higher uptake in certain innate immune cells, but not in other tested cell types, than PEGylated nanoparticles. No noticeable cytotoxicity was observed. Serum proteins did not mediate the high cell uptake of HEP-coated nanoparticles. Our work suggests that HEP polymers may be an effective surface modification technology for nanomedicines to safely and efficiently target certain innate immune cells.

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.

The biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues. By combining two-dimensional infrared spectroscopy experiments with molecular dynamics simulations, we find that calcium specifically binds to hyaluronan at millimolar concentrations. Because of its large size and charge, the calcium cation can bind simultaneously to the negatively charged carboxylate group and the amide group of adjacent saccharide units. Molecular dynamics simulations and single-chain force spectroscopy measurements provide evidence that the binding of the calcium ions weakens the intramolecular hydrogen-bond network of hyaluronan, increasing the flexibility of the polymer chain. We also observe that the binding of calcium to hyaluronan saturates at a maximum binding fraction of similar to 10-15 mol %. This saturation indicates that the binding of Ca2+ strongly reduces the probability of subsequent binding of Ca2+ at neighboring binding sites, possibly as a result of enhanced conformational fluctuations and/or electrostatic repulsion effects. Our findings provide a detailed molecular picture of ion condensation and reveal the severe effect of a few, selective and localized electrostatic interactions on the rigidity of a polyelectrolyte chain.

The biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well-recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues. By combining two-dimensional infrared spectroscopy experiments with molecular dynamics simulations, we find that calcium specifically binds to hyaluronan at millimolar concentrations. Because of its large size and charge, the calcium cation can bind simultaneously to the negatively charged carboxylate group and the amide group of adjacent saccharide units. Molecular dynamics simulations and single-chain force spectroscopy measurements provide evidence that the binding of the calcium ions weakens the intra-molecular hydrogen-bond network of hyaluronan, increasing the flexibility of the polymer chain. We also observe that the binding of calcium to hyaluronan saturates at a maximum binding fraction of ~10-15 mol %. This saturation indicates that the binding of Ca2+ strongly reduces the probability of subsequent binding of Ca2+ at neighboring binding sites, possibly as a result of enhanced conformational fluctuations and/or electrostatic repulsion effects. Our findings provide a detailed molecular picture of ion condensation, and reveal the severe effect of a few, selective and localized electrostatic interactions on the rigidity of a polyelectrolyte chain.TOC

Nanoparticles from natural and anthropogenic sources are abundant in the environment, thus human exposure to nanoparticles is inevitable. Due to this constant exposure, it is critically important to understand the potential acute and chronic adverse effects that nanoparticles may cause to humans. In this review, we explore and highlight the current state of nanotoxicology research with a focus on mechanistic understanding of nanoparticle toxicity at organ, tissue, cell, and biomolecular levels. We discuss nanotoxicity mechanisms, including generation of reactive oxygen species, nanoparticle disintegration, modulation of cell signaling pathways, protein corona formation, and poly(ethylene glycol)-mediated immunogenicity. We conclude with a perspective on potential approaches to advance current understanding of nanoparticle toxicity. Such improved understanding may lead to mitigation strategies that could enable safe application of nanoparticles in humans. Advances in nanotoxicity research will ultimately inform efforts to establish standardized regulatory frameworks with the goal of fully exploiting the potential of nanotechnology while minimizing harm to humans.

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

Many injectable drugs require delivery strategies for enhancing their pharmacokinetics due to rapid loss via renal filtration if possess low molecular weight (

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.

Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.

Unnatural chemically modified nucleotide sugars UDP-4-N(3)-GlcNAc and UDP-4-N(3)-GalNAc were chemically synthesized for the first time. These unnatural UDP sugar products were then tested for incorporation into hyaluronan, heparosan, or chondroitin using polysaccharide synthases. UDP-4-N(3)-GlcNAc served as a chain termination substrate for hyaluronan or heparosan synthases; the resulting 4-N(3)-GlcNAc-terminated hyaluronan and heparosan were then successfully conjugated with Alexa Fluor 488 DIBO alkyne, demonstrating that this approach is generally applicable for labeling and detection of suitable glycosaminoglycans.

Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ∼40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2-HS2-FGFR2 ternary complex model and the asymmetric FGF2-HS1-FGFR2 ternary complex model. In the symmetric FGF2-HS2-FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2-HS1-FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2-HS2-FGFR2 ternary complex model.

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.

Testosteronan, an unusual glycosaminoglycan (GAG) first isolated from the microbe Comamonas testosteroni, was enzymatically synthesized in vitro by transferring uridine diphosphate sugars on β-p-nitrophenyl glucuronide acceptor. After chemically converting testosteronan to N-sulfotestosteronan it was tested as a substrate for sulfotransferases involved in the biosynthesis of the GAG, heparan sulfate. Studies using (35) S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) showed that only 6-O-sulfotransferases acted on N-sulfotestosteronan. An oxidative depolymerization reaction was explored to generate oligosaccharides from (34) S-labeled 6-O-sulfo-N-sulfotestosteroran using (34) S-labeled PAPS because testosteronan was resistant to all of the tested GAG-degrading enzymes. Liquid chromotography-mass spectrometric analysis of the oxidatively depolymerized polysaccharides confirmed the incorporation of (34) S into ∼14% of the glucosamine residues. Nuclear magnetic resonance spectroscopy also showed that the sulfo groups were transferred to ∼20% of the 6-hydroxyl groups in the glucosamine residue of N-sulfotestosteronan. The bioactivity of 6-O-sulfo-N-sulfotestosteronan was examined by performing protein-binding studies with fibroblast growth factors and antithrombin (AT) III using a surface plasmon resonance competition assay. The introduction of 6-O-sulfo groups enhanced N-sulfotestosteronan binding to the fibroblast growth factors, but not to AT III.

Glycosaminoglycans (GAGs) are complex polysaccharides composed of hexosamine-containing disaccharide repeating units. The three most studied classes of GAGs, heparin/heparan sulfate, hyaluronan and chondroitin/dermatan sulfate, are essential macromolecules. GAGs isolated from animal and microbial sources have been utilized therapeutically, but naturally occurring GAGs are extremely heterogeneous limiting further development of these agents. These molecules pose difficult targets to construct by classical organic syntheses due to the long chain lengths and complex patterns of modification by sulfation and epimerization. Chemoenzymatic synthesis, a process that employs exquisite enzyme catalysts and various defined precursors (e.g. uridine 5′-diphosphosphate-sugar donors, sulfate donors, acceptors and oxazoline precursors), promises to deliver homogeneous GAGs. This review covers both theoretical and practical issues of GAG oligosaccharide and polysaccharide preparation as single molecular entities and in library formats. Even at this early stage of technology development, nearly monodisperse GAGs can be made with either natural or artificial structures.

Glycosaminoglycans (GAGs) are known to be present in all animals as well as some pathogenic microbes. Chondroitin sulfate is the most abundant GAG in mammals where it has various structural and adhesion roles. The Gram-negative bacteria Pasteurella multocida Type F and Escherichia coli K4 produce extracellular capsules composed of unsulfated chondroitin or a fructosylated chondroitin, respectively. Such polysaccharides that are structurally related to host molecules do not generally provoke a strong antibody response thus are thought to be employed as molecular camouflage during infection. We observed a sequence from the photosynthetic green sulfur bacteria, Chlorobium phaeobacteroides DSM 266, which was very similar (~62% identical) to the open reading frames of the known bifunctional chondroitin synthases (PmCS and KfoC); some segments are strikingly conserved amongst the three proteins. Recombinant E. coli-derived Chlorobium enzyme preparations were found to possess bona fide chondroitin synthase activity in vitro. This new catalyst, CpCS, however, has a more promiscuous acceptor usage than the prototypical PmCS, which may be of utility in novel chimeric GAG syntheses. The finding of such a similar chondroitin synthase enzyme in C. phaeobacteroides is unexpected for several reasons including (a) a free-living nonpathogenic organism should not "need" an animal self molecule for protection, (b) the Proteobacteria and the green sulfur bacterial lineages diverged ~2.5-3 billion years ago and (c) the ecological niches of these bacteria are not thought to overlap substantially to facilitate horizontal gene transfer. CpCS provides insight into the structure/function relationship of this class of enzymes.

A small library of well defined heparan sulfate (HS) polysaccharides was chemoenzymatically synthesized and used for a detailed structure-activity study of fibroblast growth factor (FGF) 1 and FGF2 signaling through FGF receptor (FGFR) 1c. The HS polysaccharide tested contained both undersulfated (NA) domains and highly sulfated (NS) domains as well as very well defined non-reducing termini. This study examines differences in the HS selectivity of the positive canyons of the FGF12-FGFR1c2 and FGF22-FGFR1c2 HS binding sites of the symmetric FGF2-FGFR2-HS2 signal transduction complex. The results suggest that FGF12-FGFR1c2 binding site prefers a longer NS domain at the non-reducing terminus than FGF22-FGFR1c2. In addition, FGF22-FGFR1c2 can tolerate an HS chain having an N-acetylglucosamine residue at its non-reducing end. These results clearly demonstrate the different specificity of FGF12-FGFR1c2 and FGF22-FGFR1c2 for well defined HS structures and suggest that it is now possible to chemoenzymatically synthesize precise HS polysaccharides that can selectively mediate growth factor signaling. These HS polysaccharides might be useful in both understanding and controlling the growth, proliferation, and differentiation of cells in stem cell therapies, wound healing, and the treatment of cancer.

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 Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (∼65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.

Glycosaminoglycans (GAGs) are linear hexosamine-containing polysaccharides. These polysaccharides are synthesized by some pathogenic bacteria to form an extracellular coating or capsule. This strategy forms the basis of molecular camouflage since vertebrates possess naturally occurring GAGs that are essential for life. A recent sequence database search identified a putative protein from the opportunistic pathogen Comamonas testosteroni that exhibits similarity with the Pasteurella multocida GAG synthase PmHS1, which is responsible for the synthesis of a heparosan polysaccharide capsule. Initial supportive evidence included glucuronic acid (GlcUA)-containing polysaccharides extracted from C. testosteroni KF-1. We describe here the cloning and analysis of a novel Comamonas GAG synthase, CtTS. The GAG produced by CtTS in vitro consists of the sugars d-GlcUA and N-acetyl-D-glucosamine, but is insensitive to digestion by GAG digesting enzymes, thus has distinct glycosidic linkages from vertebrate GAGs. The backbone structure of the polysaccharide product [-4-D-GlcUA-α1,4-D-GlcNAc-α1-](n) was confirmed by nuclear magnetic resonance. Therefore, this novel GAG, testosteronan, consists of the same sugars as the biomedically relevant GAGs heparosan (N-acetyl-heparosan) and hyaluronan but may have distinct properties useful for future medical applications.

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C(5)-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.

Heparosan (-GlcUA-beta1,4-GlcNAc-alpha1,4-)(n) is a member of the glycosaminoglycan polysaccharide family found in the capsule of certain pathogenic bacteria as well as the precursor for the vertebrate polymers, heparin and heparan sulfate. The two heparosan synthases from the Gram-negative bacteria Pasteurella multocida, PmHS1 and PmHS2, were efficiently expressed and purified using maltose-binding protein fusion constructs. These relatively homologous synthases displayed distinct catalytic characteristics. PmHS1, but not PmHS2, was able to produce large molecular mass (100-800 kDa) monodisperse polymers in synchronized, stoichiometrically controlled reactions in vitro. PmHS2, but not PmHS1, was able to utilize many unnatural UDP-sugar analogs (including substrates with acetamido-containing uronic acids or longer acyl chain hexosamine derivatives) in vitro. Overall these findings reveal potential differences in the active sites of these two Pasteurella enzymes. In the future, these catalysts should allow the creation of a variety of heparosan and heparinoids with utility for medical applications.

Heparin and its low molecular weight heparin derivatives, widely used as clinical anticoagulants, are acidic polysaccharide members of a family of biomacromolecules called glycosaminoglycans (GAGs). Heparin and the related heparan sulfate are biosynthesized in the Golgi apparatus of eukaryotic cells. Heparin is a polycomponent drug that currently is prepared for clinical use by extraction from animal tissues. A heparin pentasaccharide, fondaparinux, has also been prepared through chemical synthesis for use as a homogenous anticoagulant drug. Recent enabling technologies suggest that it may now be possible to synthesize heparin and its derivatives enzymatically. Moreover, new technologies including advances in synthetic carbohydrate synthesis, enzyme-based GAG synthesis, micro- and nano-display of GAGs, rapid on-line structural analysis, and microarray/microfluidic technologies might be applied to the enzymatic synthesis of heparins with defined structures and exhibiting selected activities. The advent of these new technologies also makes it possible to consider the construction of an artificial Golgi to increase our understanding of the cellular control of GAG biosyntheses in this organelle.

Poly(L-lactic acid) (PLLA) is widely used in tissue-engineering applications because of its degradation characteristics and mechanical properties, but it possesses an inert nature, affecting cell-matrix interactions. It is desirable to modify the surface of PLLA to create biomimetic scaffolds that will enhance tissue regeneration. We prepared a functionally flexible, biomimetic scaffold by derivatizing the surface of PLLA foams into primary amines, activated pyridylthiols, or sulfhydryl groups, allowing a wide variety of modifications. Poly(L-lysine) (polyK) was physically entrapped uniformly throughout the scaffold surface and in a controllable fashion by soaking the foams in an acetone-water mixture and later in a polyK solution in dimethylsulfoxide. Arginine-glycine-aspartic acid-cysteine (RGDC) adhesion peptide was linked to the polyK via creating disulfide bonds introduced through the use of the linker N-succinimidyl-3-(2-pyridylthiol)-propionate. Presence of RGDC on the surface of PLLA 2-dimensional (2-D) disks and 3-D scaffolds increased cell surface area and the number of adherent mesenchymal stem cells. We have proposed a methodology for creating biomimetic scaffolds that is easy to execute, flexible, and nondestructive.

Heparosan synthase 1 (PmHS1) from Pasteurella multocida Type D is a dual action glycosyltransferase enzyme that transfers monosaccharide units from uridine diphospho (UDP) sugar precursors to form the polysaccharide heparosan (N-acetylheparosan), which is composed of alternating (-alpha4-GlcNAc-beta1,4-GlcUA-1-) repeats. We have used molecular genetic means to remove regions nonessential for catalytic activity from the amino- and the carboxyl-terminal regions as well as characterized the functional regions involved in GlcUA-transferase activity and in GlcNAc-transferase activity. Mutation of either one of the two regions containing aspartate-X-aspartate (DXD) residue-containing motifs resulted in complete or substantial loss of heparosan polymerizing activity. However, certain mutant proteins retained only GlcUA-transferase activity while some constructs possessed only GlcNAc-transferase activity. Therefore, it appears that the PmHS1 polypeptide is composed of two types of glycosyltransferases in a single polypeptide as was found for the Pasteurella multocida Type A PmHAS, the hyaluronan synthase that makes the alternating (-beta3-GlcNAc-beta1,4-GlcUA-1-) polymer. However, there is low amino acid similarity between the PmHAS and PmHS1 enzymes, and the relative placement of the GlcUA-transferase and GlcNAc-transferase domains within the two polypeptides is reversed. Even though the monosaccharide compositions of hyaluronan and heparosan are identical, such differences in the sequences of the catalysts are expected because the PmHAS employs only inverting sugar transfer mechanisms whereas PmHS1 requires both retaining and inverting mechanisms.

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 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-π 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 [13C, 15N]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 β-glucuronidase and β-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 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.

Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating beta3N-acetylglucosamine (GlcNAc)-beta4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1-117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686-703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (beta3GalNAc-beta4GlcUA) capsule. A chimeric enzyme consisting of residues 1-427 of pmHAS and residues 421-704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1-420 of pmCS and residues 428-703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225-265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.

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.

Glycosaminoglycans, a class of linear polysaccharides composed of repeating disaccharide units containing a hexosamine, are important carbohydrates found in many organisms. Vertebrates utilize glycosaminoglycans in structural, recognition, adhesion, and signaling roles. Certain pathogenic bacteria produce extracellular capsules composed of glycosaminoglycans or glycosaminoglycan-like polymers that enhance the microbes' ability to infect or to colonize the host. In the period from 1993 to 2001, bacterial enzymes were discovered that catalyze the polymerization of the repeating unit of hyaluronan, chondroitin, or N-acetylheparosan (unsulfated, unepimerized heparin). Depending on the specific carbohydrate and the microorganism, either a dual-action enzyme (synthase) that transfers two distinct monosaccharides or a pair of single-action transferases are utilized to synthesize the glycosaminoglycan polymer. Current views on the enzymology, structures, potential evolution, and the roles of the known glycosyltransferases from Streptococcus, Pasteurella, and Escherichia are discussed.

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.

Since we first reported (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184) the cloning of the hyaluronan (HA) synthase from Streptococcus pyogenes (spHAS), numerous membrane-bound HA synthases have been discovered in both prokaryotes and eukaryotes. The HASs are unique among enzymes studied to date because they mediate 6-7 discrete functions in order to assemble a polysaccharide containing hetero-disaccharide units and simultaneously effect translocation of the growing HA chain through the plasma membrane. To understand how the relatively small spHAS performs these various functions, we investigated the topological organization of the protein utilizing fusion analysis with two reporter enzymes, alkaline phosphatase and beta-galactosidase, as well as several other approaches. From these studies, we conclude that the NH2 terminus and the COOH terminus, as well as the major portion of a large central domain are localized intracellularly. The first two predicted membrane domains were confirmed to be transmembrane domains and give rise to a very small extracellular loop that is inaccessible to proteases. Several regions of the large internal central domain appear to be associated with, but do not traverse, the membrane. Following the central domain, there are two additional transmembrane domains connected by a second small extracellular loop that also is inaccessible to proteases. The COOH-terminal approximately 25% of spHAS also contains a membrane domain that does not traverse the membrane and may contain extensive re-entrant loops or amphipathic helices. Numerous membrane associations of this latter COOH-terminal region and the central domain may be required to create a pore-like structure through which a growing HA chain can be extruded to the cell exterior. Based on the high degree of similarity among Class I HAS family members, these enzymes may have a similar topological organization for their spHAS-related domains.

Type A Pasteurella multocida, an animal pathogen, employs a hyaluronan [HA] capsule to avoid host defenses. PmHAS, the 972-residue membrane-associated hyaluronan synthase, catalyzes the transfer of both GlcNAc and GlcUA to form the HA polymer. To define the catalytic and membrane-associated domains, pmHAS mutants were analyzed. PmHAS1-703 is a soluble, active HA synthase suggesting that the carboxyl-terminus is involved in membrane association of the native enzyme. PmHAS1-650 is inactive as a HA synthase, but retains GlcNAc-transferase activity. Within the pmHAS sequence, there is a duplicated domain containing a short motif, Asp-Gly-Ser, that is conserved among many beta-glycosyltransferases. Changing this aspartate in either domain to asparagine, glutamate, or lysine reduced the HA synthase activity to low levels. The mutants substituted at residue 196 possessed GlcUA-transferase activity while those substituted at residue 477 possessed GlcNAc-transferase activity. The Michaelis constants of the functional transferase activity of the various mutants, a measure of the apparent affinity of the enzymes for the precursors, were similar to wild-type values. Furthermore, mixing D196N and D477K mutant proteins in the same reaction allowed HA polymerization at levels similar to the wild-type enzyme. These results provide the first direct evidence that the synthase polypeptide utilizes two separate glycosyltransferase sites.

Hyaluronan (HA), a long linear polymer composed of alternating glucuronic acid and N-acetylglucosamine residues, is an essential polysaccharide in vertebrates and a putative virulence factor in certain microbes. All known HA synthases utilize UDP-sugar precursors. Previous reports describing the HA synthase enzymes from Streptococcus bacteria and mammals, however, did not agree on the molecular directionality of polymer elongation. We show here that a HA synthase, PmHAS, from Gram-negative P. multocida bacteria polymerizes the HA chain by the addition of sugar units to the nonreducing terminus. Recombinant PmHAS will elongate exogenous HA oligosaccharide acceptors to form long polymers in vitro; thus far no other HA synthase has displayed this capability. The directionality of synthesis was established definitively by testing the ability of PmHAS to elongate defined oligosaccharide derivatives. Analysis of the initial stages of synthesis demonstrated that PmHAS added single monosaccharide units sequentially. Apparently the fidelity of the individual sugar transfer reactions is sufficient to generate the authentic repeating structure of HA. Therefore, simultaneous addition of disaccharide block units is not required as hypothesized in some recent models of polysaccharide biosynthesis. PmHAS appears distinct from other known HA synthases based on differences in sequence, topology in the membrane, and putative reaction mechanism.

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.