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• W1569446107 abstract "We have characterized the hyaluronan (HA) synthase activity of the Xenopus DG42 gene product in vitro. The recombinant enzyme produced in yeast does not possess a nascent HA chain and, therefore, is an ideal model system for kinetic studies of the synthase's glycosyltransferase activity. The enzymatic rate was optimal from pH 7.6 to 8.1. Only the authentic sugar nucleotide precursors, UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc), were utilized to produce a large molecular weight polymer. UDP-glucose or the galactose epimers of the normal substrates did not substitute. The Michaelis constant, Km, of recombinant DG42 in membranes was 60 ± 20 and 235 ± 40 μm for UDP-GlcA and UDP-GlcNAc, respectively, which is comparable to values obtained previously from membranes derived from vertebrate cells. The apparent energy of activation for HA elongation is about 15 kilocalories/mol. DG42 polymerizes HA at average rates of about 80 to 110 monosaccharides/s in vitro. The resulting HA polysaccharide possessed molecular weights spanning 2 × 106-107 Da, corresponding to about 104 sugar residues. This is the first report characterizing a defined eukaryotic enzyme that can produce a glycosaminoglycan. We have characterized the hyaluronan (HA) synthase activity of the Xenopus DG42 gene product in vitro. The recombinant enzyme produced in yeast does not possess a nascent HA chain and, therefore, is an ideal model system for kinetic studies of the synthase's glycosyltransferase activity. The enzymatic rate was optimal from pH 7.6 to 8.1. Only the authentic sugar nucleotide precursors, UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc), were utilized to produce a large molecular weight polymer. UDP-glucose or the galactose epimers of the normal substrates did not substitute. The Michaelis constant, Km, of recombinant DG42 in membranes was 60 ± 20 and 235 ± 40 μm for UDP-GlcA and UDP-GlcNAc, respectively, which is comparable to values obtained previously from membranes derived from vertebrate cells. The apparent energy of activation for HA elongation is about 15 kilocalories/mol. DG42 polymerizes HA at average rates of about 80 to 110 monosaccharides/s in vitro. The resulting HA polysaccharide possessed molecular weights spanning 2 × 106-107 Da, corresponding to about 104 sugar residues. This is the first report characterizing a defined eukaryotic enzyme that can produce a glycosaminoglycan. Glycosaminoglycans (GAG), 1The abbreviations used are: GAG, glycosaminoglycan; HA, hyaluronic acid, hyaluronan, hyaluronate; HAS, hyaluronan synthase; GlcA, glucuronic acid; GlcNAc,N-acetylglucosamine; Glc, glucose; GalA, galacturonic acid; GalNAc, N-acetylgalactosamine; DTT, dithiothreitol; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2–2(hydroxymethyl)-propane1,3-diol.1The abbreviations used are: GAG, glycosaminoglycan; HA, hyaluronic acid, hyaluronan, hyaluronate; HAS, hyaluronan synthase; GlcA, glucuronic acid; GlcNAc,N-acetylglucosamine; Glc, glucose; GalA, galacturonic acid; GalNAc, N-acetylgalactosamine; DTT, dithiothreitol; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2–2(hydroxymethyl)-propane1,3-diol. linear polysaccharides based on a repeating disaccharide that usually consists of an amino sugar and a negatively charged sugar, are essential constituents of higher animals. Hyaluronan (HA), heparin, and chondroitan, dermatan, and keratan sulfates are members of this class of carbohydrates. HA (→4)-β-d-GlcA(1→3)-β-d-GlcNAc(1→) is a prominent GAG that plays roles as a structural element and a recognition molecule in vertebrates (1Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Google Scholar). The enzymes that catalyze the production of HA, the HA synthases, were the first glycosyltransferases capable of forming the disaccharide repeat of a GAG to be cloned and described at the molecular level. The initial HA synthase to be identified was HasA of Streptococcus pyogenes which is the enzyme responsible for the formation of an extracellular capsule of HA in this human bacterial pathogen (2DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 14568-14571Google Scholar, 3DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Google Scholar). The HasA protein is strongly associated with the phospholipid membrane and is predicted to possess 4 or 5 membrane-spanning segments (3DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Google Scholar, 4DeAngelis P.L. Yang N. Weigel P.H. Biochem. Biophys. Res. Commun. 1994; 199: 1-10Google Scholar). The enzyme utilizes UDP-GlcA and UDP-GlcNAc precursors found in the cytosol and extrudes the growing HA chain out of the cell during polymerization. HasA, a single protein, transfers both GlcA and GlcNAc residues to HA based on genetic and biochemical evidence (3DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Google Scholar, 5DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Google Scholar). A Xenopus laevis (African clawed frog) protein, DG42 (fordifferentially expressed in gastrulation), with a previously unknown function (6Rosa F. Sargent T.D. Rebbert M.L. Michaels G.S. Jamrich M. Grunz H. Jonas E. Winkles J.A. Dawid I.B. Dev. Biol. 1988; 129: 114-123Google Scholar) was found to be quite similar at the amino acid sequence level to the bacterial HasA enzyme (3DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Google Scholar, 4DeAngelis P.L. Yang N. Weigel P.H. Biochem. Biophys. Res. Commun. 1994; 199: 1-10Google Scholar) as well as fungal chitin synthases (7Atkinson E.M. Long S.R. Mol. Plant-Microbe Interact. 1992; 5: 439-442Google Scholar). These observations led to the hypothesis that this vertebrate protein was also a HA synthase (4DeAngelis P.L. Yang N. Weigel P.H. Biochem. Biophys. Res. Commun. 1994; 199: 1-10Google Scholar, 7Atkinson E.M. Long S.R. Mol. Plant-Microbe Interact. 1992; 5: 439-442Google Scholar). DG42 contains predicted transmembrane segments clustered at both the amino and carboxyl termini; this positioning is similar to that of the membrane-associated regions found in HasA (4DeAngelis P.L. Yang N. Weigel P.H. Biochem. Biophys. Res. Commun. 1994; 199: 1-10Google Scholar). DG42 was subsequently shown to be involved in HA biosynthesis by overexpression studies. Infection of mammalian cells with a recombinant vaccinia virus construct containing the DG42 cDNA directed these cells to produce more HA than the uninfected host cells alone or vector-infected cells (8Meyer M.F. Kreil G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4543-4547Google Scholar). Definitive proof that DG42 was a bona fide HA synthase was obtained through overexpression studies in Saccharomyces cerevisiae, an eukaryotic host that doesnot normally make the HA polysaccharide. Yeast with the cloned DG42 cDNA on an expression plasmid produced a functional HA synthase (9DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Google Scholar). The recombinant enzyme transferred both GlcA and GlcNAc residues from UDP-sugar nucleotide donors to form a high molecular weight polymer. This material was degraded by the specific HA lyase from Streptomyces (9DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Google Scholar), an enzyme that does not digest any other GAG (10Ohya T. Kaneko Y. Biochim. Biophys. Acta. 1970; 198: 607-609Google Scholar). The resulting fragments from the yeast-derived polymer were identical to those generated from authentic vertebrate HA as deemed by high performance liquid chromatography analysis (9DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Google Scholar). The HA synthase activity of the recombinant yeast was localized to the membrane fraction in agreement with both the predictions derived from the DG42 primary sequence and the previous characterizations of the HA synthase from mammalian sources. In 1996, at least four reports were made of mammalian homologs possessing ∼50% identity to the DG42 protein (11Itano N. Kimata K. J. Biol. Chem. 1996; 271: 9875-9878Google Scholar, 12Spicer A.P. Augustine M.L. McDonald J.A. J. Biol. Chem. 1996; 271: 23400-23406Google Scholar, 13Shyjan A.M. Heldin P. Butcher E.C. Yoshino T. Briskin M.J. J. Biol. Chem. 1996; 271: 23395-23399Google Scholar, 14Watanabe K. Yamaguchi Y. J. Biol. Chem. 1996; 271: 22945-22948Google Scholar). Two of these reports utilized polymerase chain reaction and degenerate primers based on the hasA and DG42 sequences to obtain their clones (12Spicer A.P. Augustine M.L. McDonald J.A. J. Biol. Chem. 1996; 271: 23400-23406Google Scholar, 14Watanabe K. Yamaguchi Y. J. Biol. Chem. 1996; 271: 22945-22948Google Scholar). The cDNAs corresponding to these homologs, when overexpressed on recombinant plasmids, substantially increased HA production of transfected mammalian cells in comparison to the host cells' basal levels. It appears that at least three putative hyaluronan synthases encoded by three separate but related genes, namedHAS1, HAS2, and HAS3, exist in human and mouse. 2A. P. Spicer, personal communication.2A. P. Spicer, personal communication. The Xenopus DG42 gene is most closely related to mammalian HAS1based upon conservation of exon/intron boundaries.2 In this report, we have characterized the requirements and kinetics of recombinant DG42 produced in yeast. All reagents were from Sigma unless noted otherwise. The construction and the use of the DG42 expression plasmid for studies in yeast were described by DeAngelis and Achyuthan (9DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Google Scholar). Briefly, the DG42 cDNA, encoding a polypeptide of 588 residues, was cloned into the pYES2 vector (Invitrogen) under control of the GAL1 promoter to form pYES/DG+. Upon induction with galactose, active DG42 accumulated in the plasma membrane fraction. Membranes were prepared by the same glass-bead disruption protocol except for three alterations: (i) the more soluble and stable protease inhibitor aminoethylbenzenesulfonyl fluoride was substituted for phenylmethanesulfonyl fluoride; (ii) the repeated freeze-thawing cycles were omitted; and (iii) some preparations were lysed utilizing a MiniBeadbeater-8 (Biospec). Preparations with about 10-fold higher specific activity than our previous report were obtained when all of these modifications were utilized. Protein was quantitated by the Coomassie dye-binding assay (15Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) with a bovine serum albumin standard (Pierce). A DNA fragment encoding the open reading frame of 419 residues corresponding to streptococcal HasA (original Val codon switched to Met; Ref. 3DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Google Scholar) was also subcloned by standard methods into the pYES2 yeast expression vector to produce pYES/HA. Membranes from cells with this construct were prepared in the same fashion as pYES/DG+. The samples derived from pYES/HA constructs contained substantial HA synthase activity and a unique 42-kDa protein could be detected on Western blots with antibodies against HasA; membranes from cells with vector alone possessed neither activity nor the immunoreactive band (not shown). The incorporation of sugars into high molecular weight HA polysaccharide was monitored using UDP-[14C]GlcA (291 mCi/mmol; ICN) and/or UDP-[3H]GlcNAc (27.3 Ci/mmol; NEN Life Science Products Inc.) precursors as described previously (9DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Google Scholar). For determining optimal reaction conditions, the membrane preparations were incubated at 30 °C for 1 h, unless noted otherwise, in a buffer typically containing: 50 mm buffer ion, 0–30 mm divalent metal ion, 1 mm dithiothreitol (DTT), 0–150 μm UDP-GlcA, and 0–300 μm UDP-GlcNAc. Reactions were terminated by the addition of SDS to 2% (w/v). Descending paper chromatography (65:35, ethanol, 1 mammonium acetate, pH 5.5) was utilized to separate products from substrates; the radioactive polymers at the origin of the paper chromatogram were detected by liquid scintillation counting. Assays for characterization of the kinetic optima of DG42 were set so that <5% of the radiolabeled substrate was consumed and the enzyme concentration was in the linear range. For determining the temperature dependence of DG42 activity, 360 μm UDP-GlcA and 1 mm UDP-GlcNAc were employed in 30-min assays to obtain maximal velocity measurements. For the sugar nucleotide specificity studies, one of the authentic HA precursors was substituted with a closely related structural analog.Km values for the substrates were obtained by holding one radiolabeled UDP-sugar at a constant and saturating concentration while titrating the other UDP-sugar. The data were analyzed by graphing on Hanes-Woolf plots. Membranes (385 μg of protein) were incubated with 400 μmUDP-[14C]GlcA (1 μCi) and 900 μmunlabeled UDP-GlcNAc in 50 mm Tris, pH 7.6, 20 mm MgCl2, and 1 mm DTT (550 μl reaction volume) at 30 °C and samples (100 μl) of the reaction mixture were withdrawn at various times. The synthase was inactivated by the addition of SDS to 0.5% and the samples were deproteinized by Pronase® treatment (0.5 mg/ml final, overnight at 37 °C; Boehringer Mannheim). A parallel study with membranes containing yeast-derived recombinant HasA was performed as above except that the buffer was pH 7.0. The unincorporated precursors and small molecules (≤3 × 103 Da) were removed by ultrafiltration (3 buffer changes with a Microcon® 3 unit; Amicon). After clarification by centrifugation (16,000 × g, 5 min), one-third of the sample was injected onto a Sephacryl S-500HR gel filtration column (Pharmacia: 1 × 51 cm, 40 ml) equilibrated in 0.2 mNaCl, 5 mm Tris, pH 8. The column was eluted at 0.5 ml/min and radioactivity in the fractions (1 ml) was quantitated by liquid scintillation counting after adding EconoSafe mixture (4.5 ml, Research Products Int.). The Mr of the HA chains was calculated using the linear relationship of the Kav to log Mr(Kav = (Ve −Vo)/(Vt −Vo), where Ve is elution volume;Vo is void volume; and Vt is the total column volume) (16Determan H. Gel Chromatography. Springer-Verlag, Inc., New York1968Google Scholar). The column was calibrated with blue dextran 2000 (Pharmacia, average ∼2 × 106 Da), the only available carbohydrate standard that elutes in the linear range of the column. The size of dextran molecules excluded from Sephacryl S-500HR beads was estimated by the manufacturer (using defined microsphere standards and extrapolation) to be ∼2 × 107 Da. Even if the excluded size was actually 1.5 × 107 Da, however, our polymerization rates would be only 10% lower because the HA peaks used for the rate determinations eluted with midrange or higher Kav values. We found that the transport and the availability of sugar nucleotides across the phospholipid membranes were not the rate-limiting steps with our yeast preparations. Similar HA polymerization experiments using treatments that permeabilize lipid vesicles (preincubation with 0.05% (w/v) digitonin (final) or a pore-forming protein, perfringolysin O (1 μg/150 μg of membrane protein; generously provided by R. Tweten, University of Oklahoma)) did not alter the chromatography profiles from experiments using untreated membranes (data not shown). The DG42 enzyme in membranes was assayed under various conditions to determine the optimal pH, metal ion concentration, and ionic strength for HA polymerization. The enzyme displayed a pH optima around neutrality and the highest activities were observed in Tris-based buffers at pH 7.6 to 8.1 (Fig. 1). The synthase retained ∼80% of maximal activity from pH 7.0 to 8.4. The enzyme activity was linear for at least 2 h at pH 7.6 (data not shown). The enzyme did not perform as well in phosphate buffer; in this case, the phosphate ion probably chelates a substantial proportion of the required Mg2+ ion (data not shown). We explored the possibility that metal ions other than Mg2+could substitute as a cofactor for HA polymerization using reactions buffered at pH of 7.6. No other metal ion, including Mn2+, Co2+, Cu2+, or Ni2+, was as effective as Mg2+ (Fig. 2). No more than 13 or 23% of the incorporation observed for 20 mm Mg2+ was detected when Ni2+ or Mn2+, respectively, were substituted at the same concentration. The dependence of HA polymerization on ionic strength was measured by addition of NaCl to reactions at pH 7.6 containing 20 mmMg2+ (data not shown). The Tris buffer and Mg2+alone contribute an ionic strength of 0.11 molal. The activity remained fairly constant up to ∼0.4 molal. At higher ionic strengths, the activity gradually diminished and reached 15% of the maximal activity at 1.3 molal. DG42 was assayed at various temperatures from 0 °C to 70 °C with limiting enzyme (Fig. 3). The enzyme was not active when assayed at ≤4 °C. DG42 exhibited a roughly linear response with respect to temperature from 30 °C to 42 °C. The activity at 42 °C was twice as great as that measured at 30 °C. At 50 °C, only ∼20% of maximal activity was observed, and DG42 was completely inactive when assayed at 75 °C. The initial velocity data from reactions at 30 °C to 42 °C were plotted versus the reciprocal of temperature on an Arrhenius plot (not shown). The slope yielded a value of ∼15 kcal/mol for the apparent energy of activation, Ea, for the glycosyltransferase reactions. The Km of DG42 for the substrates UDP-GlcA and UDP-GlcNAc was determined by measuring synthase activity as a function of UDP-sugar concentration. We obtainedKm values of 60 ± 20 and 235 ± 40 μm for UDP-GlcA and UDP-GlcNAc, respectively (Figs.4 and5).Figure 5Hanes-Woolf plot estimation of Km for UDP-sugars precursors of DG42. The specific incorporation data used to generate Fig. 4, A(UDP-GlcNAc, ▪) and B (UDP-GlcA, •), were graphed as [S]/v versus [S]. The x axis intercept, which signifies −Km, yielded the Kmvalues of 235 ± 40 and 60 ± 20 μm for UDP-GlcNAc and UDP-GlcA, respectively.View Large Image Figure ViewerDownload (PPT) We examined if UDP-sugars other than UDP-GlcA and UDP-GlcNAc, the natural HA precursors, could be polymerized by DG42 (Table I). The galactose analogs, UDP-GalA and UDP-GalNAc, which are C-4 epimers of the normal substrates, could not substitute. UDP-Glc, without the C-6 carboxyl or the C-2 deoxy acetamido group of UDP-GlcA and UDP-GlcNAc, respectively, could not be polymerized in place of the natural substrates. In addition to measuring radioactivity at the origin of the paper chromatograms, where high molecular weight HA is typically found, samples were also taken at positions between the origin and the peak of the unincorporated precursors. No difference in the level of radioactivity was observed among any of the assays with various precursors. This finding suggests that no appreciable amounts of smaller polymer chains (e.g.3–6 sugars), which could possibly migrate away from the origin in our solvent system, were formed with the tested unnatural precursors.Table ISugar nucleotide specificity of recombinant DG42 hyaluronan synthaseSecond sugar nucleotide present[14C]GlcA dpm[3H]GlcNAc dpm%None90 (0.3)3501-aThe small amount of radioactive material at the origin in this reaction was chitin polysaccharide produced by the endogenous chitin synthase activity; the material was susceptible to degradation by chitinase 63 (provided by P. Robbins), and it was also formed by control membranes derived from cells with vector or antisense plasmids under similar reaction conditions. (2.2)UDP-GlcAND1-bND, not determined.16,100 (100)UDP-GlcNAc30,300 (100)NDUDP-Glc80 (0.3)240 (1.6)UDP-GalAND320 (2.0)UDP-GalNAc70 (0.2)ND1-a The small amount of radioactive material at the origin in this reaction was chitin polysaccharide produced by the endogenous chitin synthase activity; the material was susceptible to degradation by chitinase 63 (provided by P. Robbins), and it was also formed by control membranes derived from cells with vector or antisense plasmids under similar reaction conditions.1-b ND, not determined. Open table in a new tab We estimated the average HA polymerization rate by incubating the HASs in reactions with saturating concentrations of precursors (as determined in the Km studies) for defined times and determining the HA product size by gel filtration. Only data from early time points was utilized to assure that a single round of HA elongation was being observed. The rate was calculated by dividing the average chain length of the polymer peak by the duration of the HAS reaction. Recombinant yeast-derived DG42 enzyme is particularly useful in these experiments because there are no endogenous HA chains on the freshly isolated enzyme; S. cerevisiae does not produce UDP-GlcA, which is a required precursor of HA (9DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Google Scholar). This is in contrast to HA synthases isolated from vertebrate cells which contain partially formed or completed HA chains. High molecular weight HA of 2–4 ×106Da was formed by DG42 within 2.5 min (Fig. 6 A), corresponding to an average polymerization rate of 110 ± 30 monosaccharides/s. After 5 min, DG42 produced molecules of at least 3–7 × 106Da which corresponds to an average rate of 80 ± 30 monosaccharides/s. The peak of radiolabeled HA gradually increased in both molecular weight and amplitude over time. Using the data in the central portion of the HA peak after a 45-min reaction (∼50% of the total incorporation was in these fractions), we estimate that the average molecular weight of the final product of DG42 is about 9 ± 3 × 106 Da. However, some chains (∼20% of total) are ≥2 × 107 Da since they eluted in the void volume of the Sephacryl S-500 column (Fig. 6 C). In parallel experiments, streptococcal HasA produced material at an average rate of 60 ± 20 monosaccharides/min, which is almost as rapid as DG42 (Fig. 6 B), but the final product had a higher average molecular weight (≥2 × 107 Da; Fig. 6 C). Before the advent of the recombinant enzymes, several groups had studied the native HA synthase(s) derived from various vertebrate cell lines. The enzymes' requirements and Km for the UDP-sugar substrates were measured (17Ishimoto N. Temin H.M. Strominger J.L. J. Biol. Chem. 1966; 241: 2052-2057Google Scholar, 18Appel A. Horwitz A.L. Dorfman A. J. Biol. Chem. 1979; 254: 12199-12203Google Scholar, 19Philipson L.H. Schwartz N.B. J. Biol. Chem. 1984; 259: 5017-5023Google Scholar, 20Ng K.F. Schwartz N.B. J. Biol. Chem. 1989; 264: 11776-11783Google Scholar, 21Malinowski N.M. Cysyk R.L. August E.M. Biochem. Mol. Biol. Int. 1995; 35: 1123-1132Google Scholar). There were potential complications with these studies because (i) multiple HA synthase isozymes exist, and (ii) the enzymes isolated from mammalian cells possess partially elongated nascent chains and/or completed HA chains. We have utilized the yeast expression system to circumvent these pitfalls. First, since yeast do not normally make HA, the activity of a cloned synthase can be analyzed without the contributions of other endogenous HA synthases that are found in the mammalian systems. Second, yeast is a host which does not form the required UDP-GlcA precursor of HA in vivo, therefore the recombinant enzyme produced in this system cannot make a HA chain until the precursor is added to the isolated membrane preparations. This feature facilitates analysis of HA biosynthesis since all polymerization occurs de novo. In particular, the controversy surrounding the direction of HA polymer growth may be answered in the near future utilizing the yeast system. Most other known carbohydrates are synthesized by the addition of the new saccharide residue from an activated sugar nucleotide to the nonreducing end of the nascent chain. In contrast, the current model advocated by Prehm (22Prehm P. Biochem. J. 1983; 211: 181-189Google Scholar, 23Prehm P. Biochem. J. 1983; 211: 191-198Google Scholar) is that HA is polymerized by transfer of sugars to the reducing end of the molecule. One line of evidence that led to this hypothesis was that the nascent HA polymers synthesized by mammalian cell membranes apparently contain a covalently attached UDP moiety (23Prehm P. Biochem. J. 1983; 211: 191-198Google Scholar). All of the HA synthases described to date, from Gram-positiveStreptococcus and Gram-negative Pasteurellabacteria and from vertebrates, including Xenopus, utilize UDP-sugar nucleotide precursors at neutral pH (9DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Google Scholar, 17Ishimoto N. Temin H.M. Strominger J.L. J. Biol. Chem. 1966; 241: 2052-2057Google Scholar, 18Appel A. Horwitz A.L. Dorfman A. J. Biol. Chem. 1979; 254: 12199-12203Google Scholar, 19Philipson L.H. Schwartz N.B. J. Biol. Chem. 1984; 259: 5017-5023Google Scholar, 20Ng K.F. Schwartz N.B. J. Biol. Chem. 1989; 264: 11776-11783Google Scholar, 21Malinowski N.M. Cysyk R.L. August E.M. Biochem. Mol. Biol. Int. 1995; 35: 1123-1132Google Scholar, 24Markovitz A. Cifonelli J.A. Dorfman A. J. Biol. Chem. 1959; 234: 2343-2350Google Scholar, 25DeAngelis P.L. Biochem. 1996; 35: 9768-9771Google Scholar). These HA synthases require a divalent metal ion to function, but the enzymes display different preferences in vitro. Mg2+ supports the highest efficiency polymerization of HA for all enzymes tested to date, except for Pasteurellaenzyme for which 1 mm Mn2+ serves ∼2-fold better than 10 mm Mg2+ (25DeAngelis P.L. Biochem. 1996; 35: 9768-9771Google Scholar). The temperature dependence of HA synthase activity of DG42 suggests that the apparent Ea, the energy of activation for elongating HA, is ∼15 kcal/mol. This value is similar to that observed for a wide spectrum of other biosynthetic enzymes. If the transfer of one of the sugar groups, either GlcA or GlcNAc, to the HA chain had a greater energy barrier, then our calculated apparentEa value would be a reflection of the reaction with the higher activation energy. However, the GlcA and GlcNAc groups are both transferred by a UDP donor and both of the resulting glycosidic bonds are β-linked. Therefore, the actual Eavalues may be very similar for both reactions catalyzed by HAS. To make the proper assumptions concerning the similarities in reactive encounters or transition states during catalysis, it would be useful to know if the HASs possess (i) a common binding site that interacts with both UDP-sugars or (ii) two distinct binding sites for UDP-GlcA and UDP-GlcNAc. DG42 displays a greater affinity for the UDP-GlcA precursor than UDP-GlcNAc, a characteristic of all other known HA synthases. We found that the Km values of recombinant DG42 in yeast for the precursors were higher but quite similar in magnitude to values (UDP-GlcA, 3–50 μm; UDP-GlcNAc, 21–100 μm) reported by others for the membrane-associated enzyme derived from adult human, murine, or chicken cells (17Ishimoto N. Temin H.M. Strominger J.L. J. Biol. Chem. 1966; 241: 2052-2057Google Scholar, 18Appel A. Horwitz A.L. Dorfman A. J. Biol. Chem. 1979; 254: 12199-12203Google Scholar, 19Philipson L.H. Schwartz N.B. J. Biol. Chem. 1984; 259: 5017-5023Google Scholar, 20Ng K.F. Schwartz N.B. J. Biol. Chem. 1989; 264: 11776-11783Google Scholar, 21Malinowski N.M. Cysyk R.L. August E.M. Biochem. Mol. Biol. Int. 1995; 35: 1123-1132Google Scholar). The variance in Km values may be due to the intrinsic characteristics among the different HA synthase isozymes, but further analysis will be required to resolve if this disparity is just a matter of different source species, purification protocols, or assay methods. Yeast-derived DG42 exhibited exquisite specificity for the authentic sugar nucleotide precursors of the HA polysaccharide. The galactose epimers and UDP-Glc could not substitute for UDP-GlcA and UDP-GlcNAc. Similarly, streptococcal HasA only incorporated the authentic precursors into polymer (5DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Google Scholar). This selectivity suggests that the enzyme needs to make several critical contacts with the substrates, including the substituents at C-2 and C-4 of GlcNAc, and the substituents at C-4 and C-6 of GlcA. We estimated the average rates of HA polymerization by measuring the length of the HA chains produced in vitro by gel filtration and then dividing the average size by the reaction time. Our method of calculating the product size is limited by several factors: (i) the experimental difficulty in separating polymers in the 106-108 Da range with existing aqueous-based chromatography media; (ii) the relative lack of defined high molecular weight standards; and (iii) the intrinsic polydisperse nature of polysaccharides. To combat the imprecision of the gel filtration estimates of Mr, we used data from sequential reaction times and calculated the size across the median fractions of the HA peak. We presented conservative estimates of the polymerization rate based on the average size of the product molecules, but some of the HA chains are longer than the average value. Therefore, the enzyme may elongate HA at even higher rates. Our parallel studies indicate that both yeast-derived recombinant DG42 and HasA polymerize HA rapidly. Interestingly, comparison of the gel filtration profiles reveals that the ultimate size of the HA products from the two enzymes are different. DG42 produces HA polysaccharide with a smaller average size (6–12 × 106 Da) than that formed by HasA (≥2 × 107 Da). The HA size distribution produced by DG42, nonetheless, is comparable to high quality HA isolated from vertebrate tissues (1Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Google Scholar). Overall, the findings in this report substantiate yeast as a useful expression system for studies of HA synthases. Apparently no post-translational modifications unique to vertebrates, and absent in Saccharomyces, are required for correct enzymatic function of DG42, a putative HAS1-type vertebrate synthase. Furthermore, since yeast-derived HasA also functioned very well as a HA synthase, no other bacterial-specific components for HA synthesis are required; this is further evidence that one glycosyltransferase can indeed utilize two distinct substrates. It will be interesting to examine other vertebrate HA synthases to determine if the enzymological characteristics of a particular synthase isozyme customize its function in different tissues of the body and/or at various times during development. A comparison of the enzymology of the other GAG glycosyltransferases, yet to be molecularly cloned, to the HA synthases should also prove illuminating in a mechanistic as well as evolutionary sense. We thank Drs. Paul H. Weigel and Pierre Neuenschwander for helpful discussions. We also thank Dr. Andrew P. Spicer for sharing preliminary information concerning the multiple vertebrate HAS genes." @default.
• W1569446107 created "2016-06-24" @default.
• W1569446107 creator A5030464076 @default.
• W1569446107 creator A5049760871 @default.
• W1569446107 creator A5063205323 @default.
• W1569446107 date "1998-02-01" @default.
• W1569446107 modified "2023-09-30" @default.
• W1569446107 title "Enzymological Characterization of Recombinant XenopusDG42, A Vertebrate Hyaluronan Synthase" @default.
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