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

Wei Jing and Paul L. DeAngelis1

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104, USA

Received on January 27, 2000; revised on March 27, 2000; accepted on March 27, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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 ß-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.

Key words: capsule/glycosyltransferase/hyaluronan/polysaccharide/synthase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Glycosyltransferases are enzymes that catalyze the addition of monosaccharides to acceptor groups. Numerous varieties of transferases form carbohydrates ranging from oligosaccharides to polysaccharides and form glycoconjugates including glycoproteins, proteoglycans, and a myriad of natural products. One of the early dogmas of glycobiology was that one glycosyltransferase protein transfers only one specific sugar, the "one enzyme, one linkage" hypothesis of Hagopian and Eylar (1968)Go. However, several enzymes consisting of a single type of polypeptide chain have been demonstrated or suspected to catalyze the transfer of at least two distinct sugars. Perhaps the most documented examples are the hyaluronan synthases [HASs] that polymerize a polysaccharide composed of repeating disaccharide ß1->4GlcUAß1->3GlcNAc units (Weigel et al., 1997Go; DeAngelis, 1999aGo). The HAS polypeptides from certain pathogenic bacteria, vertebrates, and an algal virus have been shown to be both selective ß-GlcNAc-transferases and ß-GlcUA-transferases by molecular genetic and/or biochemical methods. Transformation of a HAS gene on a plasmid into foreign hosts that normally do not synthesize HA conferred the recombinant cells with the ability to produce HA polymer in vivo (streptococcal HAS in Escherichia coli or Enterococcus faecalis; DeAngelis et al., 1993aGo,b) or their extracts to synthesize HA in vitro (vertebrate HAS in Saccharomyces cerevisiae; DeAngelis and Achyuthan, 1996Go; viral HAS in E.coli, DeAngelis et al., 1997Go). Immunopurified streptococcal HAS protein (DeAngelis and Weigel, 1994Go) or mouse HAS1 protein (Yoshida et al., 2000Go) produced HA in vitro when supplied with the appropriate UDP-sugar precursors.

Two classes of HA synthase have been proposed on the basis of sequence similarities, predicted topology, and putative reaction mechanism (DeAngelis, 1999aGo). Certain potential sequence motifs of 5–14 amino acid residues in length are similar among the Group A and C Streptococcus bacteria, vertebrate, and viral HA synthases (Class I). The unique HAS from Type A Pasteurella multocida bacteria (Class II) has a set of motifs that are more similar to sequences found in other glycosyltransferases that make other bacterial capsular polysaccharides or lipopolysaccharides, but possesses two motifs in common with the Class I HA synthases (DeAngelis et al., 1998Go). The conserved residues are thought to be involved in catalysis or substrate binding. During the final stage of preparing this article, a report was made that a specific residue in a Class 1 motif, (S/G)GPL(G/S)xY(R/K), was associated with the ß-GlcUA-transferase catalytic activity of a HA synthase. Substitution of the leucine at position 314 of mouse HAS1 with a valine resulted in a loss of HA synthase activity, but the mutant protein could still make chitin-like GlcNAc-polymers in vitro (Yoshida et al., 2000Go). The functional role of this residue has not been assigned.

Work on the E.coli K5 enzyme KfiC that synthesizes the structurally related heparosan polysaccharide [{alpha}1->4GlcUAß1->4GlcNAc] has suggested that this protein can also transfer two different monosaccharides to the appropriate acceptor oligosaccharide (Petit et al., 1995Go). However, repeated polymerization to an acceptor chain has not been demonstrated in vitro; only one GlcNAc or one GlcUA monosaccharide can be transferred to a GlcUA-terminating or a GlcNAc-terminating oligosaccharide, respectively. Mutagenesis and deletion analysis has shown that the GlcUA-transferase activity of KfiC can be reduced or eliminated while retaining the GlcNAc-transferase activity (Griffiths et al., 1998Go).

A family of glycosyltransferases that synthesize ß-linked polysaccharides has been proposed based largely on amino acid sequence comparisons and knowledge of the transferase reactions (Saxena et al., 1995Go). Two types of domain, named "A" and "B," have been tentatively identified by hydrophobic cluster analysis. One or two of these putative domains may exist within a single polypeptide depending on the enzyme. Proteins with domains A and B appear to be associated with processive polymerization. On the other hand, nonprocessive enzymes only appear to possess domain A. These observations have led to mechanistic hypotheses invoking multiple binding sites for nucleotide-sugar precursors and simultaneous disaccharide formation by enzymes such as cellulose synthase and HA synthase (Saxena et al., 1995Go). In the case of pmHAS, we have previously found that single sugars were added individually to the nascent HA chain; the fidelity of the two transferase activities yields the disaccharide repeats of the polymer (DeAngelis, 1999bGo). In this report, we dissect the two distinct transferase activities of the pmHAS enzyme by molecular genetic means. Our results indicate that the pmHAS polypeptide contains two relatively independent transferase sites.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Deletion analysis of pmHAS
To identify the important domains of the 972-residue pmHAS polypeptide, the protein was truncated at the amino- and/or the carboxyl- termini. Polymerase chain reaction with primers corresponding to various internal sequences was used to generate a series of recombinant proteins for expression (Table I). The truncated polypeptides were expressed well in E.coli and the experimentally determined molecular weight corresponded to the predicted size (Figure 1). In vitro assays were utilized to assess the HA synthase activity, or the two half-reactions, either GlcNAc-Tase or GlcUA-Tase, that comprise HA polymerization (Table I). Some of the truncations were inactive. PmHAS1-756, which lacks the carboxyl-terminal 216 amino acid residues, was an active HA synthase and, for the most part, membrane-associated. An interesting observation was that pmHAS1-703, which lacks a larger portion of the carboxyl terminus, retained HAS activity but was transformed into a cytoplasmic protein accounting for up to ~10% of the total cellular protein. This suggested that the carboxyl-terminus, especially residues 703–756, may be responsible for the association of native pmHAS with the membrane. With the further deletion from carboxyl-terminus, pmHAS1-650 was still expressed at a high level as a soluble protein, yet was inactive as a HA synthase. However, pmHAS1-650 was capable of transferring GlcNAc to the nonreducing terminal GlcUA of HA-derived oligosaccharides. As expected from the lack of HAS activity, pmHAS1-650 did not transfer GlcUA to HA oligosaccharides, which terminated with a GlcNAc residue. This result suggested that residues 650–703 are required, either directly or indirectly, for transferring GlcUA to the HA chain. PmHAS1-567, with a further truncation at the carboxyl terminus, and pmHAS152–756 were insoluble, inactive proteins. These latter mutant proteins are likely to be misfolded inclusion bodies as they were not dissolved by a buffer containing the detergents NP-40, sodium deoxycholate and SDS unless boiled; in contrast, full-length pmHAS was readily solubilized by this buffer at room temperature.


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Table I. Apparent subcellular localization and enzyme activity of recombinant pmHAS and pmHAS truncations
 


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Fig. 1. Expression of pmHAS and its truncated forms. Either whole cell lysates (pmHAS437-972, pmHAS1-650, pmHAS1-567 and pmHAS152-756) or membrane preparations (pmHAS437–756, pmHAS1-756, r1-972, n1-972) or B-Per extract (pmHAS1-703) were analyzed by Western blot (r, recombinant from E.coli; n, native from P-1059). The bars on the left denote the position of molecular weight standards (from top to bottom: 112, 95, 55, and 29 kDa).

 
Site-directed mutagenesis of pmHAS1-703
Based on similarities in the amino acid sequence and predicted topology, two families of HASs are proposed (DeAngelis, 1999aGo). The only member of Class II, pmHAS, possesses motifs similar to two out of the seven putative conserved motifs of Class I HASs; these motifs contain DGS and DxD sequences. We observed that the pmHAS sequence has a duplication of a ~100-residue long element in the regions from residue 161–267 and from residue 443–547 with these conserved motifs. We named these two elements of pmHAS that contain the conserved motif domain A1 and domain A2, respectively. This nomenclature is based on the similarity of these pmHAS domains to the "A" domain proposed for other glycosyltransferases that make ß-linked carbohydrates (Saxena et al., 1995Go; B.Henrissat, personal communication). Figure 2 shows the amino acid alignment of the two putative domains and their relative position in pmHAS1-703. The above truncation results suggest that the GlcNAc-transferase activity could be separated from the HA synthase activity of pmHAS. Therefore, we hypothesized that domain A1 was responsible for the GlcNAc-transferase function of HA synthase while domain A2 was responsible for GlcUA-transferase activity. To test this model further, pmHAS1-703, the shortest polypeptide with complete HAS activity, was subjected to site-directed mutagenesis. We mutated the conserved aspartate residues (residue 196 and 477; Figure 2) of the two DGS motifs in the two domains.



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Fig. 2. Domains A1 and A2 of pmHAS. (A) The approximate relative positions of domain A1 and A2 in pmHAS and pmHAS1-703. (B) Partial alignment of the amino acid sequences of the two domains (residue 161–267 and 443–547). The aspartate residues mutated in our studies were marked with an asterisk. Identical residues are in bold.

 
Six different mutants were produced containing the following changes: domain A1— D196E, D196N, D196K, and domain A2—D477N, D477E, D477K. Upon sequence verification of the complete open reading frame, we found that mutants with D196K, D196N, or D477N also had spontaneous mutation of D702I. As it was the penultimate residue of pmHAS1-703, and as pmHAS1-650 was a functional GlcNAc-Tase, we did not believe that this undesired mutation would greatly affect the interpretation of the results of our desired point mutations (As the results below demonstrate, the mutants with substitutions at D196 or D477 sharing the same D702I mutation had different transferase activities supporting this assumption). All of the mutant proteins were produced at similar levels (data not shown). All of the mutants were either inactive or made long HA polymer with low efficiency as measured by the full HAS assay (Table II). However, pmHAS1-703 domain A1 mutants containing D196E, D196K, or D196N maintained high levels of GlcUA-transferase activity. On the other hand, pmHAS1-703 domain A2 mutants containing D477E, D477K, or D477N had high levels of GlcNAc-transferase activity implying that the two aspartate residues were critical for HA synthase function. This result greatly strengthened our hypothesis that two distinct transferase domains exist in the pmHAS enzyme; domain A1 is the predicted GlcNAc-transferase and domain A2 is the predicted GlcUA-transferase.


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Table II. Specific activities of various pmHAS1-703 mutants
 
KM analysis of mutants
In order to detect potential interaction or cross-talk between the two putative domains of pmHAS, we compared the apparent affinity of the wild-type and the pmHAS1-703 mutants for the UDP-GlcNAc or for the UDP-GlcUA substrates by measuring their Michaelis constants (KM) for the functional transferase activity. Titration of the UDP-sugars in the half assays for the GlcUA and GlcNAc transferases were performed (Table III). The results indicated that the KM values of the domain A1 or A2 mutants were not very different from the wild-type sequence pmHAS1-703. This finding suggests that functional disruption of one glycosyltransferase domain of pmHAS does not affect greatly the other domain.


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Table III. KM values for UDP-sugar precursors of pmHAS1-703 and mutant proteins
 
Complementation of HAS activity with two mutant proteins in vitro
We speculated that the domain A1 and the domain A2 mutants would fulfill the complete function of a HAS even if present on separate polypeptide molecules if the mutants were mixed together in the same reaction. The standard HA synthesis assay was performed with extracts containing either the truncated wild-type sequence pmHAS1-703 enzyme, or a GlcNAc-Tase mutant enzyme (D196N) alone, or a GlcUA-Tase mutant enzyme (D477K) alone, or a mixture of the two mutant enzymes. These two mutants were selected as they were the least active in the HA synthase assay (Table II). Equivalent amounts of wild-type pmHAS1-703 polypeptide (2 µg of total protein) or mutant pmHAS1-703 polypeptide (based on Western blot analysis) were used for these assays. In the mixture, the same amount of each mutant polypeptide was added (equivalent to 4 µg of total protein of wild-type extract). The D196N mutant alone or the D477K mutant alone did not produce detectable amounts of HA chains (Figure 3), but when the mutant polypeptides were incubated together, along with a HA oligosaccharide acceptor (4–10 sugars long), longer HA polymers were made. The amount and the rate of HAS activity of the combination of the two mutants was similar to the parallel reaction containing the wild-type pmHAS1-703. Without HA oligosaccharide acceptor, the wild-type pmHAS1-703 enzyme could still make HA, albeit with lower efficiency (2 µg total protein in 3 h assay incorporated 220 dpm). The combination of the two mutant extracts, however, did not make detectable amounts of HA polymer in absence of the HA acceptor (incorporation < 4 dpm). These results suggested that in the presence of HA oligosaccharide acceptor, the two kinds of transferases could work together and sequentially transfer GlcNAc and GlcUA monosaccharides to an existing HA chain in an alternating fashion. Apparently chain initiation requires two active transferases to be present on the same polypeptide.



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Fig. 3. Complementation of the HAS activity of mutant enzymes in vitro. HAS enzyme assays with HA-derived acceptor were performed in the presence of either wild type pmHAS1-703 alone, or D196 mutant alone, or D477 mutant alone or in the presence of both D196 and D477 mutants, for either 25 min (open bars) or 1.5 h (solid bars).

 
Gel filtration chromatography studies were performed to analyze the size of the HA products polymerized by reaction mixtures containing either the wild-type pmHAS1-703 or a combination of the GlcUA-transferase and the GlcNAc-transferase mutant enzymes. Our results showed that the size distribution of the HA products from either reaction were similar; polymers with an average peak size of ~28–30 kDa (~150 sugars) were detected after a 3 min incubation. Therefore, the two individual mutant transferase polypeptides worked together with almost the same efficiency as the wild-type enzyme consisting of a single polypeptide chain.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Putative membrane localization or anchor domain of pmHAS
All known native HASs are found in membrane preparations upon lysis and fractionation of cells. Class I HASs, which include the streptococcal, vertebrate and viral enzymes, have similar predicted topology with five to seven membrane-associated regions in the membrane bilayer (Weigel et al., 1997Go; DeAngelis, 1999aGo). On the other hand, pmHAS, the only member of Class II, is predicted to have two transmembrane helices by some computer analysis programs (TmPRED), while other algorithms (SOSUI) classify the enzyme as a soluble protein. In any case, the majority of the pmHAS polypeptide chain is not predicted to be associated with the membrane based on its amino acid sequence alone. After removal of the residues from 703 to 756, a membrane-associated form of pmHAS was transformed into a soluble cytoplasmic protein. The most simplistic hypothesis is that the carboxyl terminus is required to target or to bind pmHAS to the membrane (Figure 4). However, this region of pmHAS is predicted neither to be membrane-associated (TmPRED, SOSUI, and HMMTOP) nor serve as a site for a post-translational lipidation (as assessed by PROSITE) based on its sequence. Perhaps some of the residues in the region of residues 703–972 of pmHAS interact with another membrane bilayer-associated protein to mediate the localization to the membrane. Alternatively, the carboxyl terminus of pmHAS may interact with an insoluble partner that is not necessarily a membrane protein. Experiments directed at ascertaining the nature of the partner are in progress. This exploration may prove illuminating in the realm of capsular polysaccharide biosynthesis and transport in Gram-negative bacteria.



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Fig. 4. Model of the two putative glycosyltransferase sites and the potential membrane association region of the pmHAS polypeptide.

 
Role of conserved aspartate residues in the DGS motif of pmHAS
Several acidic aspartate and/or glutamate residues are conserved in the putative ß-glycosyltransferase family members which utilize nucleotide-sugars as precursors to make polysaccharides (Saxena et al., 1995Go). These carboxylate sidechains are likely to play roles as catalytic acids or bases, in precursor binding, or in complexing essential metal ions. The amino acid sequence of the pmHAS polypeptide is distinct from the sequences of Class I HASs. All known HASs, however, share the DGS sequence motif containing an aspartate residue that appears to also be conserved among other ß-glycosyltransferase family members. In the present study, after the aspartate residue of the DGS motif was mutated in domain A1 or domain A2, these pmHAS1-703 mutants almost completely lost HA synthase function by losing the ability to transfer either GlcNAc or GlcUA, respectively. The x-ray crystal structure of the putative UDP-sugar transferase SpsA of Bacillus subtilis with bound UDP (Charnock and Davies, 1999Go) became available during this study. One region of this protein (residues 1–117) has some similarity to the pmHAS A1 and A2 domains (32% and 31% identity, respectively; Multalin). Their results show that the aspartate residue in the DGS motif is involved in binding the UDP-sugar by interacting with the N3 group of the uracil ring. Unfortunately, the sugar transferase specificity of the SpsA enzyme is currently unknown. We extrapolate that our pmHAS aspartate mutants may have lost the ability to bind one of the precursor sugars and, therefore, do not incorporate the monosaccharide into the HA chain. The mutant proteins with asparagine or glutamate substitutions at D196 or D477 were inactive or very poor transferases, indicating that both the size and the charge of the aspartate side chain of this motif is very important for interaction with uracil. Experiments with purified enzymes will be required to further evaluate this hypothesis.

Two-site model of a dual glycosyltransferase for heteropolysaccharide biosynthesis
The data from the activity analyses of the truncated versions and the point mutants of pmHAS strongly suggest that two relatively independent active sites on one polypeptide are responsible for the alternating addition of GlcUA and GlcNAc monosaccharides during HA polymerization. The selective disruption of either the GlcNAc-transferase or the GlcUA-transferase does not perturb significantly the remaining transferase activity as measured by KM values. Likewise, the successful rescue of HA synthase activity in vitro by mixing two different mutants supports the two-site model. The simplest explanation for the reconstitution of HA synthase activity is that the nascent HA chain is extended by one functional transferase, released, and extended by the other transferase in a repetitive fashion.

At this time, we do not believe that two pmHAS polypeptides form a dimer to create the active HA synthase species in vivo. Preliminary data from radiation inactivation studies of native or recombinant full-length pmHAS yields a target size of ~110 kDa for the functional size of the HA synthase which corresponds to the mass of one pmHAS monomer (Pummill, Kempner, and DeAngelis, unpublished observations). An alternative model in which a single transferase site on the polypeptide is responsible for transferring both of the GlcNAc and GlcUA monosaccharides in an alternating fashion also seems much less likely in view of our mutagenesis data and the domain organization of pmHAS. The exact demarcation of the domain A1 and A2 boundaries will require further analysis by molecular biological, biochemical, and possibly, structural means.

The precise identification of the putative HA acceptor-binding site of pmHAS has not yet been defined, but as judged by the activity of the various mutants in the half-reaction assays, the site probably resides in the first 650 residues. As discussed elsewhere (DeAngelis, 1999bGo), this acceptor binding site is probably responsible for maintaining the nonreducing terminus of the nascent HA chain in close proximity to the residues involved in transferase activity. The number and nature of residues required for polymer binding awaits analysis.

Conclusions
This report demonstrates for the first time the molecular dissection of a glycosyltransferase enzyme that normally forms a repeating heteropolysaccharide into its two functional constituent transferases. In previous cases, only the GlcNAc-Tase activity of the polysaccharide synthase was preserved (Griffiths et al., 1998Go; Yoshida et al., 2000Go). We demonstrate in the case of pmHAS that the glycobiology dogma of "one transferase protein, one linkage" does not hold; a single polypeptide can contain two separate UDP-sugar binding and transferase sites. As pmHAS is rather distinct from the Class I enzymes, it is unclear as yet which lessons may be applied to the other HASs. It is likely, however, that the DGS motif is responsible in part for UDP-sugar binding in both classes of HAS. With respect to the ß-linked glycosyltransferase domain theory of Saxena et al. (1995)Go, thus far, the behavior of pmHAS, an enzyme with duplicated A domains, supports the general characteristic of domain A as a nonprocessive polymerase unit.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Reagents
Molecular biology reagents were from Promega unless noted. Custom oligonucleotides were from The Great American Gene Company. All other reagents were the highest grade available from either Sigma or Fisher unless otherwise noted.

Generation of pmHAS truncations and site-directed mutagenesis
A series of truncated polypeptides were generated by amplifying the pPm7A insert (DeAngelis et al., 1998Go) by 13 cycles of PCR with Taq polymerase (Fisher) and synthetic oligonucleotide primers corresponding to various portions of the pmHAS open reading frame. The primers contained EcoRI and PstI restriction sites to facilitate cloning into the expression plasmid pKK223–3 (tac promoter; Pharmacia). The resulting recombinant constructs were transformed into E.coli TOP 10F' cells (Invitrogen) and maintained on Luria-Bertani media with ampicillin selection. Mutations were made using the QuickChange site-directed mutagenesis method (Stratagene) with the plasmid pKK/pmHAS1-703 DNA as template. The sequences of the mutant open reading frames were verified by automated DNA sequencing (Oklahoma State University Recombinant DNA/Protein Resource Facility).

Enzyme preparation
Membrane preparations containing recombinant full length pmHAS, pmHAS437-972, pmHAS437-756, pmHAS1-756, pmHAS1-567, and pmHAS152-756 were isolated from E.coli as described (DeAngelis and Weigel, 1994Go). For soluble truncated pmHAS proteins, pmHAS1-703, pmHAS1-650, and pmHAS1-703-derived mutants, cells were extracted with B-Per II Bacterial Protein Extraction Reagent (Pierce) according to the manufacturer’s instruction except that the procedure was performed at 7°C in the presence of protease inhibitors. Membrane preparations of P.multocida P-1059 (ATCC 15742) were made as described previously (DeAngelis, 1996Go). To test whether the truncated recombinant polypeptides were formed as insoluble inclusion bodies, membrane preparations were suspended in RIPA buffer (1% NP-40, 1% sodium deoxycholate and 0.1% SDS in 50 mM Tris, pH 7.2) for 20 min at room temperature. After centrifugation at 20,000 x g for 10 min, the supernatants were saved and the pellets were resuspended in RIPA buffer. The supernatants and the pellets were analyzed by SDS-polyacrylamide gel electrophoresis and Western blot analysis as described later.

Enzyme assay for HA polymerization, GlcNAc transfer, or GlcUA transfer
Three assays were designed to detect either (1) the polymerization of long HA chains, or (2) the addition of a single GlcNAc to a GlcUA-terminated HA oligosaccharide acceptor, or (3) the addition of a single GlcUA to a GlcNAc-terminated HA oligosaccharide acceptor. The complete HAS activity was assayed in 50 mM Tris, pH 7.2, 20 mM MnCl2, 0.1 M (NH4)2SO4, 1 M ethylene glycol, 0.12 mM UDP-[14C]GlcUA (0.01 µCi; NEN), 0.3 mM UDP-GlcNAc, and even-numbered HA oligosaccharides (1 µg uronic acid) derived from testicular hyaluronidase [(GlcNAc-GlcUA)n; n = 4–10] (DeAngelis, 1999bGo) at 30°C for 25 min in a reaction volume of 50 µl. GlcNAc-transferase activity was assayed for 4 min in the same buffer system with even-numbered HA oligosaccharides but with only one precursor sugar, 0.3 mM UDP-[3H]GlcNAc (0.2 µCi; NEN). GlcUA-transferase activity was assayed for 4 min in the same buffer system but with only 0.12 mM UDP-[14C]GlcUA (0.02 µCi) and odd-numbered HA oligosaccharides [GlcNAc(GlcUA-GlcNAc)n; n = 7–20] (3.5 µg uronic acid) prepared by mercuric acetate treatment of Streptomyces HA lyase digests (Ludwigs et al., 1987Go). Reactions were terminated by the addition of SDS to 2% (w/v). The reaction products were separated from substrates by descending paper (Whatman 3M) chromatography with ethanol/1 M ammonium acetate, pH 5.5, development solvent (65:35 for the HAS and GlcUA-Tase assays; 75:25 for the GlcNAc-Tase assay). For the HAS assay, the origin of the paper strip was eluted with water and the incorporation of radioactive sugars into HA polymer was detected by liquid scintillation counting with BioSafe II cocktail (RPI). For the half-assay reactions, the origin and the downstream 6 cm of the strip was counted in 2 cm pieces. All assays were adjusted to be linear with regard to incubation time and to protein concentration. For the KM studies, the UDP-sugar concentration was titrated in the half-assay reactions (0–2000 µM UDP-GlcNAc or 0–1200 µM UDP-GlcUA) and 6-fold more HA oligosaccharide acceptor was utilized.

Western blot analysis
Membranes and extracts were analyzed using standard 8% polyacrylamide SDS gels. Following electrophoresis, proteins were transferred with a semi-dry apparatus to nitrocellulose membranes (S&S) and detected with a monospecific antibody directed against a synthetic peptide corresponding to residues 526 to 543 of pmHAS. The peptide, acetyl-LDSDDYLEPDAVELCLKE-amide (Quantum), was coupled to ovalbumin (DeAngelis and Weigel, 1994Go) to form the initial immunogen for injection into female New Zealand White rabbits (HTI Bioscience protocols). In the subsequent boosts, free peptide was utilized. The specific antipeptide IgG was purified from ammonium sulfate fractionated sera (after third boost) using an immobilized peptide column (internal cysteine coupled to Iodoacetyl beads; Pierce). The desired IgG was eluted with 0.1 M glycine, pH 2.5, neutralized, and exchanged into phosphate-buffered saline. Immunoreactive bands on Western blots were detected with a protein A-alkaline phosphatase conjugate and were visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium reagent.

Gel filtration chromatography
The size of HA polymers was analyzed by chromatography on a Phenomenex PolySep-GFC-P 3000 column (300 x 7.8 mm) eluted with 0.2 M sodium nitrate at 0.6 ml/min on a Waters 600E system. The column was standardized with various size fluorescent dextrans (580, 50, and 12 kDa). Radioactive components were detected with a LB508 Radioflow Detector (EG & G Berthold) and Zinsser cocktail (1.8 ml/min). In comparison to the full HAS assay using paper chromatography described above, these 3 min reactions contained twice the UDP-sugar concentrations, 0.06 µCi UDP-[14C]GlcUA, and 0.25 µg even-numbered HA oligosaccharide. Also, addition of ethylenediamine tetracetic acid (final conc. 22 mM) and boiling (2 min) was employed to terminate the reactions instead of addition of SDS.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Drs. Gillian Air, Richard Cummings, Paul Weigel, and Phillip Pummill for helpful comments and Annette Fleshman and Amy Padgett-McCue for technical assistance.

This work was supported by grants from the National Science Foundation (MCB-9876193) and the National Institutes of Health (R01-GM56497).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
HA, hyaluronan or hyaluronate or hyaluronic acid; HAS, hyaluronan synthase; pmHAS, Pasteurella multocida hyaluronan synthase; GlcNAc, N-acetyl-glucosamine; GlcNAc-Tase, GlcNAc transferase; GlcUA, glucuronic acid; GlcUA-Tase, GlcUA transferase; KM, Michaelis constant; SDS, sodium dodecyl sulfate; UDP-, uridine diphospho-.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Charnock,S.J. and Davies,G.J. (1999) Structure of the nucleotide-diphospho-sugar transferases, spsA from Bacillus subtilis, in native and nucleotide-complexed forms. Biochemistry, 38, 6380–6385.[ISI][Medline]

DeAngelis,P.L., Papaconstantinou,J. and Weigel,P.H. (1993a) Isolation of a Streptococcus pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria. J. Biol. Chem., 268, 14568–14571.[Abstract/Free Full Text]

DeAngelis,P.L., Papaconstantinou,J. and Weigel,P.H. (1993b) Molecular cloning, identification and sequence of the hyaluronan synthase gene from Group A Streptococcus pyogenes. J. Biol. Chem., 268, 19181–19184.[Abstract/Free Full Text]

DeAngelis,P.L. and Weigel,P.H. (1994) Immunochemical confirmation of the primary structure of streptococcal hyaluronan synthase and synthesis of high molecular weight product by the recombinant enzyme. Biochemistry, 33, 9033–9039.[ISI][Medline]

DeAngelis,P.L. (1996) Enzymological characterization of the Pasteurella multocida hyaluronan synthase. Biochemistry, 35, 9768–9771.[ISI][Medline]

DeAngelis, PL. and Achyuthan,A.M. (1996) Yeast-derived recombinant DG42 protein of Xenopus can synthesize hyaluronan in vitro. J. Biol. Chem., 271, 23657–23660.[Abstract/Free Full Text]

DeAngelis,P.L., Jing,W., Graves,M.V., Burbank,D.E. and Van Etten,J.L. (1997) Hyaluronan synthase of chlorella virus PBCV-1. Science, 278, 1800–1803.[Abstract/Free Full Text]

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