Enzymological Characterization of Recombinant XenopusDG42, A Vertebrate Hyaluronan Synthase

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. 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. 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-14CGlcA (291 mCi/mmol; ICN) and/or UDP-3HGlcNAc (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-14CGlcA (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 × the excluded size was × Da, our rates be only the HA used for the rate eluted with or higher Kav We found that the and the of sugar the phospholipid membranes were not the with our yeast HA using that with or a protein, (1 μg of membrane by of did not the chromatography from using membranes not shown). The DG42 enzyme in membranes was under various to the optimal metal and for HA polymerization. The enzyme a pH optima and the were in at pH 7.6 to The synthase of maximal activity from pH to The enzyme activity was linear for at least 2 at pH 7.6 not shown). The enzyme did not as well in in this the a substantial of the not shown). We the that metal other than as a for HA using at pH of other metal ion, or was as as more than or of the incorporation for 20 mm was detected when or respectively, were substituted at the same The dependence of HA on was by addition of to at pH 7.6 containing 20 not shown). The buffer and an of The activity constant to higher the activity and of the maximal activity at DG42 was at various from °C to °C with enzyme The enzyme was not active when at DG42 a linear with to temperature from 30 °C to The activity at °C was as as that at 30 50 only of maximal activity was and DG42 was when at The initial velocity data from at 30 °C to °C were the of temperature on an (not shown). The a of for the apparent energy of for the glycosyltransferase The of DG42 for the substrates UDP-GlcA and UDP-GlcNAc was by synthase activity as a function of UDP-sugar We values of 60 ± 20 and 235 ± 40 μm for UDP-GlcA and UDP-GlcNAc, of for precursors of The specific incorporation data used to and were as The which the of 235 ± 40 and 60 ± 20 μm for UDP-GlcNAc and UDP-GlcA, We other than UDP-GlcA and UDP-GlcNAc, the HA precursors, could be by DG42 The galactose and which are epimers of the normal could not substitute. the carboxyl or the of UDP-GlcA and UDP-GlcNAc, respectively, could not be in of the In addition to radioactivity at the origin of the paper where high molecular weight HA is typically samples were also at the origin and the of the unincorporated in the level of radioactivity was any of the assays with various This that of polymer chains which could from the origin in our were with the nucleotide specificity of recombinant DG42 hyaluronan sugar nucleotide small of radioactive material at the origin in this reaction was chitin polysaccharide produced by the chitin synthase the material was to by by P. and was also by control membranes derived from cells with vector or under similar reaction not The small of radioactive material at the origin in this reaction was chitin polysaccharide produced by the chitin synthase the material was to by by P. and was also by control membranes derived from cells with vector or under similar reaction not in a We estimated the average HA rate by the in with saturating of precursors in the for defined and determining the HA product size by gel Only data from was utilized to that a single of HA elongation was The rate was calculated by the average chain of the polymer by the of the yeast-derived DG42 enzyme is in these are HA chains on the S. does not produce UDP-GlcA, which is a of HA (9DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Google Scholar). This is in to HA synthases from vertebrate cells which or HA molecular weight HA of was by DG42 corresponding to an average rate of 110 ± 30 After 5 DG42 produced molecules of at least × which to an average rate of 80 ± 30 The of radiolabeled HA increased in both molecular weight and the data in the of the HA after a reaction of the total incorporation was in these we that the average molecular weight of the product of DG42 is about ± 3 × 106 some chains of are × eluted in the void of the Sephacryl column In parallel streptococcal HasA produced material at an average rate of 60 ± 20 which is as as DG42 but the product a higher average molecular weight × the of the recombinant the HA derived from various vertebrate cell The requirements and for the UDP-sugar substrates were N. J. Biol. Chem. Scholar, A. A. J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, E.M. Biochem. Mol. Biol. Scholar). were with these studies (i) HA synthase and (ii) the enzymes from mammalian cells possess nascent chains and/or HA We have utilized the yeast expression system to these yeast not normally make HA, the activity of a cloned synthase can be analyzed the of other HA synthases that are found in the mammalian yeast is a host which does not form the UDP-GlcA of HA in the recombinant enzyme produced in this system make a HA chain the is to the membrane This analysis of HA biosynthesis all In the the of HA polymer be in the utilizing the yeast other are by the addition of the from an sugar nucleotide to the of the nascent In the model by P. Biochem. J. Scholar, P. Biochem. J. Scholar) is that HA is by of sugars to the of the of evidence that led to this hypothesis was that the nascent HA polymers by mammalian cell membranes a P. Biochem. J. Scholar). All of the HA synthases described to from and and from UDP-sugar nucleotide precursors at pH (9DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Google Scholar, N. J. Biol. Chem. Scholar, A. A. J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, E.M. Biochem. Mol. Biol. Scholar, A. J.A. A. J. Biol. Chem. Scholar, P.L. Biochem. 1996; Scholar). These HA synthases a divalent metal to but the enzymes in vitro. the of HA for all enzymes to except for for which 1 mm than mm P.L. Biochem. 1996; Scholar). The temperature dependence of HA synthase activity of DG42 that the apparent the energy of activation for HA, is This is similar to that for a of other the of one of the sugar GlcA or to the HA chain a energy our calculated be a of the reaction with the higher activation the GlcA and GlcNAc are both transferred by a and both of the resulting are the be similar for both by make the the in or during be to the possess (i) a that with both or (ii) for UDP-GlcA and UDP-GlcNAc. DG42 a for the UDP-GlcA than UDP-GlcNAc, a of all other HA We found that the values of recombinant DG42 in yeast for the precursors were higher but quite similar in to values UDP-GlcNAc, by for the membrane-associated enzyme derived from or cells N. J. Biol. Chem. Scholar, A. A. J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, E.M. Biochem. Mol. Biol. Scholar). The in values be to the the HA synthase but analysis be to this is a of or assay DG42 specificity for the authentic sugar nucleotide precursors of the HA polysaccharide. The galactose epimers and could not for UDP-GlcA and UDP-GlcNAc. streptococcal HasA only the authentic precursors into polymer P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Google Scholar). This that the enzyme to make with the the at and of and the at and of We estimated the average rates of HA by the of the HA chains produced in by gel filtration and the average size by the reaction of the product size is by (i) the in polymers in the range with chromatography (ii) the of defined high molecular weight and (iii) the of the of the gel filtration of we used data from reaction and calculated the size the fractions of the HA We of the rate based on the average size of the product but some of the HA chains are than the average the enzyme HA at higher parallel studies that both yeast-derived recombinant DG42 and HasA HA comparison of the gel filtration that the size of the HA products from the enzymes are DG42 HA polysaccharide with a average size × 106 Da) than that by HasA × The HA size produced by is comparable to high HA from vertebrate (1Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Google Scholar). the in this report yeast as a expression system for studies of HA modifications unique to and in are for enzymatic function of a putative vertebrate yeast-derived HasA also well as a HA other for HA are this is evidence that one glycosyltransferase can It be to other vertebrate HA synthases to the of a synthase function in of the and/or at various during A comparison of the of the other GAG to be to the HA synthases also in a as well as We H. Weigel and for We also P. for the vertebrate