From the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
![]() |
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.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glycosaminoglycans
(GAG),1 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)-
-D-GlcA(1
3)-
-D-GlcNAc(1
) is
a prominent GAG that plays roles as a structural element and a
recognition molecule in vertebrates (1). 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 (2, 3). The HasA protein is strongly associated with the phospholipid membrane and is predicted to possess 4 or 5 membrane-spanning segments (3, 4). 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 (3, 5).
A Xenopus laevis (African clawed frog) protein, DG42 (for differentially expressed in gastrulation), with a previously unknown function (6) was found to be quite similar at the amino acid sequence level to the bacterial HasA enzyme (3, 4) as well as fungal chitin synthases (7). These observations led to the hypothesis that this vertebrate protein was also a HA synthase (4, 7). 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 (4). 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 (8). Definitive proof that DG42 was a bona fide HA synthase was obtained through overexpression studies in Saccharomyces cerevisiae, an eukaryotic host that does not normally make the HA polysaccharide. Yeast with the cloned DG42 cDNA on an expression plasmid produced a functional HA synthase (9). 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 (9), an enzyme that does not digest any other GAG (10). 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 (9). 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 (11-14). Two of these reports utilized polymerase chain reaction and degenerate primers based on the hasA and DG42 sequences to obtain their clones (12, 14). 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, named HAS1, HAS2, and HAS3, exist in human and mouse.2 The Xenopus DG42 gene is most closely related to mammalian HAS1 based upon conservation of exon/intron boundaries.2 In this report, we have characterized the requirements and kinetics of recombinant DG42 produced in yeast.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Production of Recombinant DG42 and HasA Enzyme-- 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 (9). 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 (15) 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. 3) 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).Polysaccharide Synthase Assays-- 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 (9). 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 M ammonium 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.Analysis of HA Polymerization Rate--
Membranes (385 µg of
protein) were incubated with 400 µM
UDP-[14C]GlcA (1 µCi) and 900 µM
unlabeled 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 M
NaCl, 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) (16). 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Buffer Optima for DG42 HA Synthase Activity-- 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).
|
|
Determination of the Energy of Activation--
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.
|
Km Determination-- 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 obtained Km values of 60 ± 20 and 235 ± 40 µM for UDP-GlcA and UDP-GlcNAc, respectively (Figs. 4 and 5).
|
|
Sugar Nucleotide Specificity of DG42-- 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.
|
Analysis of Polymerization Rate--
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 (9). 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 ×106
Da was formed by DG42 within 2.5 min (Fig.
6A), corresponding to an
average polymerization rate of 110 ± 30 monosaccharides/s. After
5 min, DG42 produced molecules of at least 3-7 × 106
Da 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. 6C). In parallel
experiments, streptococcal HasA produced material at an average rate of
60 ± 20 monosaccharides/min, which is almost as rapid as DG42
(Fig. 6B), but the final product had a higher average
molecular weight (
2 × 107 Da; Fig.
6C).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (17-21). 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 (22, 23) 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 (23).
All of the HA synthases described to date, from Gram-positive Streptococcus and Gram-negative Pasteurella bacteria and from vertebrates, including Xenopus, utilize UDP-sugar nucleotide precursors at neutral pH (9, 17-21, 24, 25). 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 Pasteurella enzyme for which 1 mM Mn2+ serves ~2-fold better than 10 mM Mg2+ (25).
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 apparent
Ea 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 -linked. Therefore, the actual Ea values 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 (17-21). 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 (5). 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 (1).
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.
![]() |
ACKNOWLEDGEMENTS |
---|
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.
![]() |
FOOTNOTES |
---|
* This work was supported by a Medical Research Scholar grant from the University of Oklahoma Medical Alumni Association and National Institutes of Health Grant R01-GM56497 (to P. L. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 405-271-2227;
Fax: 405-271-3092; E-mail: Paul-DeAngelis{at}OUHSC.edu.
1 The 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.
2 A. P. Spicer, personal communication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|