Publications

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

  DeAngelis P.L.

  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 H ... >> More

  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. << Less

  J Biol Chem 274:26557-26562(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of the purified hyaluronan synthase from Streptococcus equisimilis.

  Tlapak-Simmons V.L., Baron C.A., Weigel P.H.

  Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg(2+) to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nu ... >> More

  Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg(2+) to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP, and UTP. Purified seHAS was approximately 40% more active in 25 mM, compared to 50 mM, PO(4) in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5-11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pK(a) values at pH 6.6 and 11.8. As the pH was increased from approximately 6.5, both K(m) and V(max) increased until pH approximately 10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/s) was essentially unchanged from pH 6.5 to 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Delta9) fatty acids. The apparent E(act) for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 kg/mol caused 50% inhibition of V(max) at 21, 6.5, and 3.5 mM, respectively. The apparent K(i) values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3, and 1.2 mM. << Less

  Biochemistry 43:9234-9242(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

  Itano N., Sawai T., Yoshida M., Lenas P., Yamada Y., Imagawa M., Shinomura T., Hamaguchi M., Yoshida Y., Ohnuki Y., Miyauchi S., Spicer A.P., McDonald J.A., Kimata K.

  Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyalu ... >> More

  Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions. << Less

  J. Biol. Chem. 274:25085-25092(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Molecular cloning, identification, and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes.

  DeAngelis P.L., Papaconstantinou J., Weigel P.H.

  The hyaluronan (HA) synthase of Group A Streptococci has been identified by transposon mutagenesis and deletion analysis. The genes for the HA synthase and a recently identified UDP-Glc dehydrogenase (Dougherty, B. A., and van de Rijn, I. (1993) J. Biol. Chem. 268, 7118-7124) reside on a contiguou ... >> More

  The hyaluronan (HA) synthase of Group A Streptococci has been identified by transposon mutagenesis and deletion analysis. The genes for the HA synthase and a recently identified UDP-Glc dehydrogenase (Dougherty, B. A., and van de Rijn, I. (1993) J. Biol. Chem. 268, 7118-7124) reside on a contiguous stretch of 3.2-kilobase pair DNA that can direct HA biosynthesis in Enterococcus faecalis and Escherichia coli as well as mutant Streptococcus (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 14568-14571). The synthase contains 395 residues (calculated Mr = 45,063) and migrates on SDS-PAGE with a molecular mass of 42 kDa. E. coli K5, which synthesizes UDP-glucuronic acid for production of its endogenous capsular polysaccharide, can make HA if it contains a plasmid encoding the intact 42-kDa protein. E. coli SURE or chi 1448 cells containing the same construct, however, cannot produce HA since these strains cannot make both required sugar nucleotide precursors. The HA synthase is predicted to be an integral membrane protein with four membrane-associated helices, which is consistent with the location of the enzyme activity in Streptococci. There is significant homology between the HA synthase and the Rhizobium nodC gene product, an enzyme that synthesizes chitin-like oligomers. This is the first description at the molecular level of an enzyme shown to synthesize a glycosaminoglycan. << Less

  J. Biol. Chem. 268:19181-19184(1993) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family.

  Spicer A.P., McDonald J.A.

  The three mammalian hyaluronan synthase (HAS) genes and the related Xenopus laevis gene, DG42, belong to a larger evolutionarily conserved vertebrate HAS gene family. We have characterized additional vertebrate HAS genes from chicken (chas2 and chas3) and Xenopus (xhas2, xhas3, and a unique Xenopu ... >> More

  The three mammalian hyaluronan synthase (HAS) genes and the related Xenopus laevis gene, DG42, belong to a larger evolutionarily conserved vertebrate HAS gene family. We have characterized additional vertebrate HAS genes from chicken (chas2 and chas3) and Xenopus (xhas2, xhas3, and a unique Xenopus HAS-related sequence, xHAS-rs). Genomic structure analyses demonstrated that all vertebrate HAS genes share at least one exon-intron boundary, suggesting that they evolved from a common ancestral gene. Furthermore, the Has2 and Has3 genes are identical in structure, suggesting that they arose by a gene duplication event early in vertebrate evolution. Significantly, similarities in the genomic structures of the mouse Has1 and Xenopus DG42 genes strongly suggest that they are orthologues. Northern analyses revealed a similar temporal expression pattern of HAS genes in developing mouse and Xenopus embryos. Expression of mouse Has2, Has3, and Xenopus Has1 (DG42) led to hyaluronan biosynthesis in transfected mammalian cells. However, only mouse Has2 and Has3 expressing cells formed significant hyaluronan-dependent pericellular coats in culture, implying both functional similarities and differences among vertebrate HAS enzymes. We propose that vertebrate hyaluronan biosynthesis is regulated by a comparatively ancient gene family that has arisen by sequential gene duplication and divergence. << Less

  J. Biol. Chem. 273:1923-1932(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase: two active sites exist in one polypeptide.

  Jing W., DeAngelis P.L.

  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 domain ... >> More

  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. << Less

  Glycobiology 10:883-889(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.