Chemoenzymatic synthesis with the hyaluronan synthase; production of a multitude of defined authentic, derivatized, and analog polymers.

Hyaluronan (HA; [-3-GlcNAc-1-beta-4-GlcA-1-beta] ), an essential matrix polysaccharide of vertebrates and the molecular camouflage coating in certain pathogens, is polymerized by "HA synthase" (HAS) enzymes. Three HAS classes have been identified with biotechnological utility, but only the Class II PmHAS from Type A has been useful for preparation of very defined HA polymers in vitro. Two general chemoenzymatic strategies with different size products are possible: (1) repetitive step-wise extension reactions by sequential addition of a single monosaccharide from a donor UDP-sugar onto an acceptor (or "primer") comprised of a short glycosaminoglycan chain (e.

High-fidelity and iterative affinity extraction of hyaluronan.

The glycosaminoglycan hyaluronan (HA) serves a variety of crucial physiological functions in vertebrates. Synthesized at the plasma membrane and secreted into the extracellular environment, HA polymers span a wide range of molecular weights (MW) that define their activity through a notable size-function relationship. Analytical technologies for determining HA MW distributions typically require selective extraction from complex biofluids or tissues.

CSPG4-dependent cytotoxicity for TcdB is influenced by extracellular calcium and chondroitin sulfate.

Unlabelled: TcdB is an intracellular bacterial toxin indispensable to infections. The ability to use chondroitin sulfate proteoglycan 4 (CSPG4) as a primary cell surface receptor is evolutionarily conserved by the two major variants of TcdB. As CSPG4 does not typically undergo receptor-mediated endocytosis, we sought to identify environmental factors that stabilize interactions between TcdB and CSPG4 to promote cell binding and entry into the cytosol.

Targeted Analysis of the Size Distribution of Heavy Chain-Modified Hyaluronan with Solid-State Nanopores.

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge.

Hyaluronan synthases; mechanisms, myths, & mysteries of three types of unique bifunctional glycosyltransferases.

Hyaluronan (HA), the essential [-3-GlcNAc-1-β-4-GlcA-1-β-]n matrix polysaccharide in vertebrates and molecular camouflage coating in select pathogens, is polymerized by "HA synthase" (HAS) enzymes. The first HAS identified three decades ago opened the window for new insights and biotechnological tools. This review discusses current understanding of HA biosynthesis, its biotechnological utility, and addresses some misconceptions in the literature.

Quantifying Intracellular Nanoparticle Distributions with Three-Dimensional Super-Resolution Microscopy.

Super-resolution microscopy can transform our understanding of nanoparticle-cell interactions. Here, we established a super-resolution imaging technology to visualize nanoparticle distributions inside mammalian cells. The cells were exposed to metallic nanoparticles and then embedded within different swellable hydrogels to enable quantitative three-dimensional (3D) imaging approaching electron-microscopy-like resolution using a standard light microscope.

Chemoenzymatic synthesis of sulfur-linked sugar polymers as heparanase inhibitors.

Complex carbohydrates (glycans) are major players in all organisms due to their structural, energy, and communication roles. This last essential role involves interacting and/or signaling through a plethora of glycan-binding proteins. The design and synthesis of glycans as potential drug candidates that selectively alter or perturb metabolic processes is challenging.

Controlling Nanoparticle Uptake in Innate Immune Cells with Heparosan Polysaccharides.

We used heparosan (HEP) polysaccharides for controlling nanoparticle delivery to innate immune cells. Our results show that HEP-coated nanoparticles were endocytosed in a time-dependent manner by innate immune cells via both clathrin-mediated and macropinocytosis pathways. Upon endocytosis, we observed HEP-coated nanoparticles in intracellular vesicles and the cytoplasm, demonstrating the potential for nanoparticle escape from intracellular vesicles.

A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces.

Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established.

Selective isolation of hyaluronan by solid phase adsorption to silica.

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.

Optimizing the sensitivity and resolution of hyaluronan analysis with solid-state nanopores.

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.

Methods for isolating and analyzing physiological hyaluronan: a review.

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.

Nanoparticle Surface Engineering with Heparosan Polysaccharide Reduces Serum Protein Adsorption and Enhances Cellular Uptake.

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.

Hyaluronic acid synthesis, degradation, and crosslinking in equine osteoarthritis: TNF-α-TSG-6-mediated HC-HA formation.

Background: TNF-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.

A primer for initiation of commercialization of glycobiological discoveries.

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.

Strong Reduction of the Chain Rigidity of Hyaluronan by Selective Binding of Ca Ions.

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.

Nanoparticle Toxicology.

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.

Label-free analysis of physiological hyaluronan size distribution with a solid-state nanopore sensor.

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.

Heparosan-coated liposomes for drug delivery.

Liposomal encapsulation is a useful drug delivery strategy for small molecules, especially chemotherapeutic agents such as doxorubicin. Doxil® is a doxorubicin-containing liposome ("dox-liposome") that passively targets drug to tumors while reducing side effects caused by free drug permeating and poisoning healthy tissues. Polyethylene glycol (PEG) is the hydrophilic coating of Doxil® that protects the formulation from triggering the mononuclear phagocyte system (MPS).

Synthesis and characterization of heparosan-granulocyte-colony stimulating factor conjugates: a natural sugar-based drug delivery system to treat neutropenia.

Many injectable drugs require delivery strategies for enhancing their pharmacokinetics due to rapid loss via renal filtration if possess low molecular weight (<60-70 kDa) and/or clearance by the body's components (e.g., proteases, antibodies, high-efficiency receptors) in their native form.

Synthesis of 4-Azido-N-acetylhexosamine Uridine Diphosphate Donors: Clickable Glycosaminoglycans.

Unnatural chemically modified nucleotide sugars UDP-4-N-GlcNAc and UDP-4-N-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-GlcNAc served as a chain termination substrate for hyaluronan or heparosan synthases; the resulting 4-N-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.

Expanding glycosaminoglycan chemical space: towards the creation of sulfated analogs, novel polymers and chimeric constructs.

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.

Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis.

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.

Identification of a chondroitin synthase from an unexpected source, the green sulfur bacterium Chlorobium phaeobacteroides.

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.