Paul L. DeAngelis

Hyaluronan Synthases: A Decade-plus of Novel Glycosyltransferases

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.

Hyaluronan synthases: fascinating glycosyltransferases from vertebrates, bacterial pathogens, and algal viruses

Abstract. 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.

Hyaluronic Acid Production in Bacillus subtilis

ABSTRACT 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.

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides

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 C5-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.

Identification of the capsular polysaccharides of Type D and F Pasteurella multocida as unmodified heparin and chondroitin, respectively

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.

Evolution of glycosaminoglycans and their glycosyltransferases: Implications for the extracellular matrices of animals and the capsules of pathogenic bacteria.

Glycosaminoglycans (linear polysaccharides with a repeating disaccharide backbone containing an amino sugar) are essential components of extracellular matrices of animals. These complex molecules play important structural, adhesion, and signaling roles in mammals. Direct detection of glycosaminoglycans has been reported in a variety of organisms, but perhaps more definitive tests for the glycosyltransferase genes should be utilized to clarify the distribution of glycosaminoglycans in metazoans. Recently, glycosyltransferases that form the hyaluronan, heparin/heparan, or chondroitin backbone were identified at the molecular level. The three types of glycosyltransferases appear to have evolved independently based on sequence comparisons and other characteristics. All metazoans appear to possess heparin/heparan. Chondroitin is found in some worms, arthropods, and higher animals. Hyaluronan is found only in two of the three main branches of chordates. The presence of several types of glycosaminoglycans in the body allows multiple communication channels and adhesion systems to operate simultaneously. Certain pathogenic bacteria produce extracellular coatings, called capsules, which are composed of glycosaminoglycans that increase their virulence during infection. The capsule helps shield the microbe from the host defenses and/or modulates host physiology. The bacterial and animal polysaccharides are chemically identical or at least very similar. Therefore, no immune response is generated, in contrast to the vast majority of capsular polymers from other bacteria. In microbial systems, it appears that in most cases functional convergent evolution of glycosaminoglycan glycosyltransferases occurred, rather than direct horizontal gene transfer from their vertebrate hosts. Anat Rec 268:317–326, 2002.

Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase.

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 polymersin 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.

Identification and Molecular Cloning of a Chondroitin Synthase from Pasteurella multocida Type F

Pasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocidaType A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1–704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of ∼103 sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [β(1,4)GlcUA-β(1,3)GalNAc] n . In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities.

Chemoenzymatic Synthesis with Distinct Pasteurella Heparosan Synthases MONODISPERSE POLYMERS AND UNNATURAL STRUCTURES

Heparosan (-GlcUA-β1,4-GlcNAc-α1,4-)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.

Glycosaminoglycan polysaccharide biosynthesis and production: today and tomorrow.

Glycosaminoglycans [GAGs] are essential heteropolysaccharides in vertebrate tissues that are also, in certain cases, employed as virulence factors by microbes. Hyaluronan [HA], heparin, and chondroitin sulfate [CS] are GAGs currently used in various medical applications and together are multi-billion dollar products thus targets for production by animal-free manufacture. By using bacteria as the source of GAGs, the pathogen’s sword may be converted into a plowshare to help avoid potential liabilities springing from the use of animal-derived GAGs including adventitious agents (e.g., prions, pathogens), antigenicity, degradation of the environment, and depletion of endangered species. HA from microbes, which have a chemical structure identical to human HA, has already been commercialized and sold at the ton-scale. Substantial progress towards microbial heparin and CS has been made, but these vertebrate polymers are more complicated structurally than the unsulfated bacterial polysaccharide precursors thus require additional processing steps. This review provides an overview of GAG structure, medical applications, microbial biosynthesis, and the state of bacterial GAG production systems. Representatives of all glycosyltransferase enzymes that polymerize the sugar chains of the three main GAGs have been identified and serve as the core technology to harness, but the proteins involved in sugar precursor formation and chain export steps of biosynthesis are also essential to the GAG production process. In addition, this review discusses future directions and potential important issues. Overall, this area is poised to make great headway to produce safer (both increased purity and more secure supply chains) non-animal GAG-based therapeutics.