Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida

Wei Jing1 and Paul L. DeAngelis2

Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104

Received on May 9, 2002; revised on May 9, 2003; accepted on May 22, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating ß3N-acetylglucosamine (GlcNAc)-ß4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1–117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686–703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (ß3GalNAc-ß4GlcUA) capsule. A chimeric enzyme consisting of residues 1–427 of pmHAS and residues 421–704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1–420 of pmCS and residues 428–703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225–265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.

Key words: capsule / chondroitin / glycosaminoglycan / glycosyltransferase / hyaluronan


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycosyltransferases are enzymes that catalyze the addition of monosaccharides from activated sugars (e.g., nucleotide sugars) to acceptor groups. An early dogma 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. Three examples of dual-action enzymes are most of the enzymes that polymerize glycosaminoglycans (GAGs), namely, hyaluronan synthases (HASs), chondroitin synthases (CSs), and heparin/heparosan synthases. HASs polymerize the hyaluronan (HA) polysaccharide composed of the repeating disaccharide ß4glucuronic acid (ß4GlcUA)-ß3N-acetylglucosamine (ß3GlcNAc) units (reviewed in Weigel et al., 1997Go; DeAngelis, 1999aGo) while CSs (or polymerases) polymerize a polysaccharide composed of repeating ß4GlcUA-ß3GalNAc units (DeAngelis and Padgett-McCue, 2000Go; Kitagawa et al., 2001Go; Ninomiya et al., 2002Go). Heparosan synthases synthesize heparosan, a polysaccharide of repeating ß4GlcUA-ß4GlcNAc units (Lind et al., 1998Go; DeAngelis and White, 2002Go). The reaction mechanisms of these dual-function enzymes are not yet clear.

Pasteurella multocida is a Gram-negative bacterial pathogen that infects both humans and beasts. Depending on the strain, this microbe often employs various GAG polysaccharide capsules to avoid host defenses and increase virulence (DeAngelis, 2002Go). These microbial GAGs are molecular mimics of vertebrate polymers and are relatively nonimmunogenic.

HA plays structural, recognition, and signaling roles in vertebrates. The capsule of Type A P. multocida is also composed of HA. This bacterial GAG is polymerized by pmHAS, the only known example of a Class II HA synthase (DeAngelis et al., 1998Go). This enzymologically distinct catalyst is not similar at the protein level to the Class I HA synthases of vertebrates. We have previously found that single monosaccharides were added individually to the nascent HA chain by pmHAS; the fidelity of the two transferase activities yields the disaccharide repeats of the polymer (DeAngelis, 1999bGo). Our studies also demonstrated that pmHAS uses two separate glycosyltransferase sites to catalyze the transfer of GlcNAc and GlcUA to form the HA polymer (Jing and DeAngelis, 2000Go). Within the pmHAS sequence, there is a pair of duplicated domains that are similar to the Domain A proposed by Saxena et al. (1995)Go based on sequence comparison of many glycosyltransferases. Both domains of pmHAS possess a short sequence motif containing DGS that is conserved among many ß-glycosyltransferases. Changing the aspartate in either motif to asparagine, glutamate, or lysine significantly reduced or eliminated the HAS activity. However, the D196 mutants possess high levels of GlcUA-transferase activity, whereas the D477 mutants maintain high levels of GlcNAc-transferase activity. This was the first direct demonstration to show that two independent glycosyltransferase sites coexist within a single polypeptide.

Chondroitin is one of the most prevalent GAG in vertebrates as well as part of the capsular polymer of Type F P. multocida, a minor fowl cholera pathogen. This bacterium produces unsulfated chondroitin (DeAngelis et al., 2002Go), but animals possess sulfated chondroitin polymers. The first CS from any source to be molecularly cloned was the P. multocida pmCS (DeAngelis and Padgett-McCue, 2000Go). The pmCS contains 965 amino acid residues and is about 90% identical to pmHAS. A soluble recombinant Escherichia coli–derived pmCS1–704 catalyzes the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro.

In this article, we analyze several conserved sequence motifs found in pmHAS and pmCS for their roles in polysaccharide biosynthesis and provide further evidence that both enzymes utilize two relatively independent glycosyltransferase sites. We also identify a region of these synthases that is important for discrimination between the UDP-GlcNAc and UDP-GalNAc substrates.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Analysis of truncated pmHAS proteins to delineate essential regions
To analyze the contribution of the amino terminal region of pmHAS, we produced various recombinant truncated polypeptides (pmHAS46–703, pmHAS72–703, pmHAS96–703, and pmHAS118–703) in E. coli. The experimentally determined molecular weights of the mutant proteins corresponded to the predicted sizes (data not shown). The truncated versions, pmHAS46–703 and pmHAS72–703, were very active HASs (Table I). The pmHAS96–703 and pmHAS118–703 were expressed at low levels compared with other constructs but were still functional HASs. Therefore, it is probable that deletion beyond residue 72 affected the overall folding efficiency of the entire polypeptide and the shorter mutants were degraded, as suggested by the observation of lower-molecular-weight immunoreactive bands derived from pmHAS118–703 on western blots. Overall, these findings suggest that the amino terminal 117 residues are not required for HAS catalytic activity.


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Table I. Enzyme activities of the truncated pmHAS proteins

 
Previously, we observed that pmHAS1–703 was a functional HAS but pmHAS1–650 lost its GlcUA-transferase activity. To further delineate the GlcUA-transferase domain within the carboxyl terminal region, we created two slightly longer mutants, pmHAS1–668 and pmHAS1–686. Both mutants were expressed well but did not polymerize HA due to the loss of GlcUA-transferase activity (Table I), suggesting that the carboxyl terminal boundary of the GlcUA-transferase resides between residues 686 and 703. We also tried to find the minimal size of truncated polypeptide with only GlcUA-transferase activity. The pmHAS428–703construct, which contains Domain A2 (see later discussion), expressed well as judged by western blotting but had neither GlcNAc-transferase nor GlcUA-transferase activity. It is possible that the amino terminal region to Domain A1, which has not yet been characterized, is also necessary for GlcUA-transfer activity and/or nascent HA chain binding. Thus, pmHAS46–160/428–703 was created. Unfortunately, this construct was not expressed as assessed by western blotting and no activity was detected even with high amounts of extract.

Site-directed mutagenesis of pmHAS1–703 to probe potential DGS and DXD sequence motifs
Others have used hydrophobic cluster analysis to identify two types of domains conserved in a variety of ß-linked glycosyltransferases that use nucleotide diphospho sugar as donors, termed Domain A and Domain B (Saxena et al., 1995Go). pmHAS has two Domain A–like regions; we therefore called the regions Domains A1 and A2 (Jing and DeAngelis, 2000Go) (Figure 1). According to the hydrophobic cluster analysis program (B. Henrissat personal communication), Domains A1 and A2 encompass residues 152–325 and 432–604, respectively. Characterization of two conserved DGS motifs in the two domains indicated that the two aspartate residues are essential for HAS activity (Jing and DeAngelis, 2000Go). We also noted the existence of a third potential DGS sequence motif in pmHAS located at position 563–565. To determine if this motif was as critical for synthase activity, like the other two DGS motifs, we mutated D563 of pmHAS1–703 into a glutamate, asparagine, or lysine residue. All of these mutants behaved like wild-type pmHAS1–703 (data not shown), indicating that this third DGS is not essential for the catalytic activity of pmHAS. This result also underscores the need for empirical testing for function of any short putative sequence motifs.



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Fig. 1. DXD motifs in predicted Domains A1 and A2. (A) The approximate relative positions of the two DXD motifs in the 972-residue pmHAS polypeptide and the two domains (A1 = 152–325 and A2 = 432–604) as predicted by hydrophobic cluster analysis and observations of internal sequence homology. (B) Alignment of the amino acid sequences of portions of the two domains (residues 161–267 of A1 and 443–547 of A2 are shown). The aspartate residues of the DXD motifs mutated in our current studies were marked with an asterisk. Identical residues of the alignment are in boldface.

 
The DXD motif is found in many glycosyltransferases (Wiggins and Munro, 1998Go). We observed that pmHAS has two DXD motifs, one in Domain A1 and another in Domain A2 (Figure 1). X-ray crystallography of the Bacillus SpsA protein/UDP complex suggested that the DXD motif is involved in binding metal ion coordinated with the beta phosphate and the ribose moiety of the UDP-sugar (Charnock and Davies, 1999Go). The involvement of the individual aspartate residues of both DXD motifs in pmHAS therefore was characterized. We mutated separately all of the four aspartate residues (residue 247, 249, 527, or 529; Figure 1) in pmHAS1–703. Mutants were produced containing the following changes in Domain A1: D247E, D247N, D247K, D249E, D249N, or D249K, and in Domain A2: D527N, D527E, D527K, D529E, D529N, or D529K. On sequence verification of the complete open reading frame, we found that mutants with D247N, D249K, D529E, and D527K also had a mutation of D702I, the penultimate residue, that did not affect HAS activity as explained previously (Jing and DeAngelis, 2000Go). All of the mutant proteins were produced at similar levels in soluble form as judged by western blotting (data not shown). In vitro assays were used to assess the HAS activity (e.g., polymerization of long HA chains) or the two half-reactions, either GlcNAc-transferase or GlcUA-transferase activity. All of the mutants were inactive as HASs except D529E, which had 10% of the wild-type relative specific activity (Table II). As predicted, the enzymes containing mutations at position 247 or 249 (Domain A1 mutants) maintained high levels of GlcUA-transferase activity. On the other hand, the enzymes containing mutations at position 527 or 529 (Domain A2 mutants) had high levels of GlcNAc-transferase activity. Therefore, all of the four aspartate residues were critical for HAS function. These results greatly strengthen our model of two distinct transferase sites in a single pmHAS polypeptide; Domain A1 is essential for GlcNAc-transferase activity and Domain A2 is essential for GlcUA-transferase activity.


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Table II. Relative specific activities of the various pmHAS1–703 DXD mutants

 
Differential metal preference of the two transferase activities
The two DXD motifs of pmHAS are predicted to be involved in binding the metal ion of the UDP-sugar/metal substrate complex based on the SpsA structure. Therefore, we examined (1) if other metal ions could rescue the mutants' activity and (2) if the two separate active sites have similar metal ion preference. HAS assays were performed in the presence of different metal ions. The presence of Mn2+, Co2+, Mg2+, or Ca2+, even at the level of 200 mM, did not convert any DXD mutant (excluding the D529E mutant with 10% relative activity) into a functional HAS (data not shown). We also performed GlcNAc-transferase or GlcUA-transferase assays with wild-type pmHAS1–703 in the presence of 20 mM Mn2+, Co2+, or Mg2+. Although in general the highest activities were obtained in the presence of Mn2+, the GlcNAc-transferase activity preferred Co2+ over Mg2+, and the GlcUA-transferase activity preferred Mg2+ over Co2+ (Table III). Similar results were obtained when assays were performed with the pmHAS1–703 mutants that have only a single transferase activity.


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Table III. Metal ion preference of the GlcNAc-transferase and the GlcUA-transferase activities of pmHAS

 
Kinetic analyses and rescue of mutant enzyme activity with high substrate concentration
Because pmHAS1–703-D529E has 10% of the HAS activity of wild-type enzyme, we compared its apparent Km values for UDP-GlcUA and for Mn2+ with the wild-type pmHAS1–703. The average apparent Km value for UDP-GlcUA of the D529E and the wild-type pmHAS enzyme is 230 ± 20 µM and 130 ± 40 µM, respectively (Figure 2A); this approximately twofold difference represents an only slightly lower binding affinity for the mutant. The average apparent Km value for Mn2+ ion of the D529E and the wild-type enzyme is 560 ± 80 µM and 110 ± 10 µM, respectively (Figure 2B); this approximately fivefold difference in metal binding represents a more significant lowering of affinity. Due to the extremely low HAS activity of other mutants, we could not obtain kinetic data with confidence using the current HAS assay methodology.



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Fig. 2. Apparent Km analysis of wild-type pmHAS and D529E mutant for UDP-GlcUA and Mn2+. HAS assays were performed as described in Materials and methods. A representative data set is shown. The Km values of D529E for UDP-GlcUA (A) and Mn2+ (B) are about twofold and fivefold higher, respectively, than wild-type pmHAS (wt). (A, incorporation of [3H]GlcNAc; B, incorporation of [14C]GlcUA).

 
As has already been discussed, D529E retained 10% activity relative to wild-type enzyme even at normal assay UDP-sugar concentrations (0.15–0.6 mM). However, if the hypothesis that DXD motifs are involved in substrate binding is true, then a mutant with very poor binding characteristics (e.g., <0.1% of activity relative to wild type) should be aided by providing higher concentration of substrate. We tested the mutant enzymes' HAS activity in the presence of extremely high concentrations of UDP-sugar substrate (up to 20 mM) to test if the polymerizing function could be rescued (Figure 3). In this series of experiments, for example, a GlcNAc-transferase (with a mutant GlcUA-transferase site) was tested with radiolabeled UDP-GlcNAc and high concentration of UDP-GlcUA (the substrate for the perturbed activity) while monitoring the formation of long HA polymer. The radiolabeled sugar is only added to the acceptor chain if the poor transferase site functions first; this assay allows the use of high precursor concentrations without lowering the specific activity of the radioactive tracer. Under these conditions, D247E and D249E mutants with conservative substitutions in Domain A1 showed moderate levels of relative HAS activity (70% and 20%, respectively, of the relative activity of wild-type enzyme). The D247N and D249N mutants have only 4% or 0% relative activity of the wild-type enzyme (Figure 3A). For mutations in Domain A2, neither D527E nor D527N mutants could be rescued, whereas D529N mutant showed 3% relative activity of wild-type enzyme (Figure 3B). To control for any potential lack of specificity, we used the structural analog of the authentic precursors, UDP-glucose, at high concentrations (up to 19 mM) in parallel experiments; rescue of activity was not observed with any mutant.



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Fig. 3. Activity of pmHAS mutants in the presence of high levels of UDP-sugar precursor substrates. All assays (30°C, 90 min) with mutants were carried out in duplicate with 40 µg of total protein and the results were averaged. (A) Reactions with Domain A1 mutants contained 0.15 mM UDP-[14C]-GlcUA (0.04 µCi) and 0–20 mM UDP-GlcNAc. For comparison, 2 µg of wild-type extract in these assay conditions yielded 29,000 dpm at 10 mM UDP-GlcNAc (not plotted). Closed squares, D247E; closed triangles, D249E; closed circles, D247N; open circles, D249N. (B) Reactions with Domain A2 mutants contained 0.3 mM UDP-[3H]-GlcNAc (0.1 µCi) and 0–20 mM UDP-GlcUA. For comparison, 2 µg of wild-type extract in these assay conditions yielded 35,000 dpm at 10 mM UDP-GlcNAc (not plotted). Closed triangles, D529N; open squares, D527N; closed squares, D527E. D529E retains 10% HAS activity with standard, lower precursor concentrations (Table II).

 
The ability to rescue the HA polymerizing ability with high substrate concentration may be a matter of compensating for the mutant's weak binding affinity. However, other mechanisms for increasing the reaction rate are also possible. Unfortunately, the effect of substrate concentration on catalysis in our case—a dual-action glycosyltransferase adding two similar sugars onto a nascent polymer chain—is complicated kinetically especially because we do not know all of the reaction steps. The ability to rescue some of these DXD mutants with high concentrations of UDP-sugars also suggests that the point mutation lesions are not causing the individual domains to be misfolded or denatured.

Site-directed mutagenesis of pmHAS1–703 to probe the WGGED sequence motif
In the pmHAS polypeptide sequence, there is a segmentsimilar to portions of mammalian UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGalNTransferases) that catalyzes the initial step for making the oligosaccharide moiety of O-linked glycoproteins. The W366GGED370 motif of pmHAS, which resides near the boundary of putative Domain A1 and Domain A2, does not exist in the sequences of other Class I HASs from Streptococcus, vertebrates, or Chlorella virus. To study the function of the WGGED motif in pmHAS, we mutated E369 or D370. Six different mutants were produced, each containing one of the following changes: E369D, E369Q, E369H, D370E, D370N, or D370K. All the mutants were expressed at comparable levels to the wild-type enzyme as judged by western blotting (data not shown). Based on the results of the HAS assays and the two half assays, mutation at either position 369 or 370 resulted in the loss of only GlcNAc-transferase activity but not the GlcUA-transferase activity (Table IV), suggesting that the WGGED motif in pmHAS is essential for GlcNAc-transferase activity.


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Table IV. Relative specific activities of the pmHAS1–703 WGGED mutants

 
We also performed HAS assays in the presence of 200 mM of Mn2+, Co2+, Ca2+, or Mg2+, but the mutants' HAS activity could not be rescued under these conditions. However, in the presence of 20 mM of UDP-GlcNAc, E369D mutant showed ~30% of the relative activity of wild-type enzyme; this same mutant had 0.1% of the relative activity to wild-type enzyme at the lower substrate concentration of 0.3 mM. Rescue was selective because the analog UDP-glucose at 19 mM did not increase activity of the E369D mutant. On the other hand, the E369Q and the D370E mutants showed no increase in activity at 20 mM UDP-GlcNAc. In all conditions, D370N mutant had no detectable HAS activity (data not shown).

In vitro reconstitution of HAS activity with two distinct mutant proteins
Previously we demonstrated that a combination of two pmHAS1–703 DGS motif mutants, D196N, a GlcUA-transferase, and D477K, a GlcNAc-transferase, would fulfill the complete function of a HAS when mixed together in the same reaction along with a HA oligosaccharide acceptor (Jing and DeAngelis, 2000Go). Here we performed the standard HAS activity assay with eight different combinations of DXD or WGGED mutants. One GlcNAc-transferase enzyme (a D527 or D529 mutant lacking GlcUA-transferase activity) and one GlcUA-transferase enzyme (a D247, D249, E370, or D369 mutant lacking GlcNAc-transferase activity) were combined in these tests. In the presence of HA oligosaccharide acceptors (4–10 sugars long), all of the combinations of enzymes synthesized HA polymer. In most cases, the relative polymerization efficiencies were close to wild-type pmHAS1–703 (~30–100%). Work in progress with immobilized mutant enzymes shows that the nascent HA chain is released transiently in vitro from one mutant enzyme before action by the second mutant (DeAngelis et al., 2003Go). These demonstrations further prove that the two independent transferase sites sequentially transfer GlcNAc and GlcUA monosaccharides to the nascent HA chain in an alternating fashion.

Sugar transferase domain swapping between pmHAS and pmCS
The CS from Type F P. multocida, pmCS, is about 90% identical to pmHAS at the protein level. The majority of sequence differences exist in the vicinity of Domain A1 of pmHAS, and the carboxyl terminal halves of the enzymes are almost identical (DeAngelis and Padgett-McCue, 2000Go). This observation is not surprising because the carboxyl terminal half of pmHAS contains Domain A2, which has the GlcUA-transferase active site; this activity would also be required for pmCS to form chondroitin polymer. We speculated that pmCS also possesses two separate transferase sites, but in this enzyme the amino terminal half is a GalNAc-transferase and the carboxyl terminal half is a GlcUA-transferase. If our model is accurate, then swapping the carboxyl terminal GlcUA-transferase site between pmHAS and pmCS would not affect their sugar polymerizing activity. On the other hand, swapping of the amino half of either pmHAS or pmCS should change the hexosamine transfer specificity. To test our hypothesis, domain swapping between pmHAS and pmCS was performed by the polymerase chain reaction (PCR) overlapping extension method (Horton et al., 1989Go). We used the active truncated versions of the synthases, pmCS1–704 and pmHAS1–703, as the starting materials for the construction. We chose residues 427/428 of pmHAS and the virtually equivalent site of pmCS, residues 420/421, as the initial splicing site based on comparisons of the amino acid sequences of pmHAS, pmCS, and other GlcNAc-transferases.

The chimeric enzyme comprised of residues 1–427 from pmHAS and residues 421–704 from pmCS (pm-AC construct) was an active HAS. The opposite combination, an enzyme consisting of residues 1–420 from pmCS and residues 428–703 from pmHAS (pm-BD construct), resulted in an active CS (Table V). This finding indicates that Domain A1 dictates hexosamine transfer specificity. Also, the source of the GlcUA-transferase Domain A2 does not affect the specificity of either the GalNAc-transferase or the GlcNAc-transferase activity. Again, the two single-action transferase sites of pmHAS and pmCS appear relatively independent.


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Table V. Activities of chimeric Pasteurella GAG synthases

 
Analysis of UDP-hexosamine transferase specificity
To define the critical regions in the amino terminal halves of pmHAS and pmCS that specify sugar substrate between UDP-GlcNAc and UDP-GalNAc, further domain swapping between pmHAS and pmCS was performed by PCR overlapping extension. The pm-FH, which possesses pmCS residues 1–258, is an active pmCS, although the majority of the chimeric protein is from pmHAS residues 266–703 (Table V). As more of the pmCS sequence is replaced by pmHAS sequence, as in pm-JL enzyme construct (which possesses pmCS residues 1–214 at the amino terminus and pmHAS residues 222–703 at the carboxyl terminus), the enzyme is converted into a catalyst with HAS activity. The conversion of GalNAc-transfer activity into GlcNAc-transfer activity indicates that the region encompassing residues 222–265 of pmHAS and probably the corresponding region of pmCS (residues 215–258) plays a critical role in the selectivity between binding and/or transferring the GalNAc and GlcNAc substrate of the UDP-sugar (Figure 4). These two monosaccharides are epimers that differ only in the orientation of a single hydroxyl group at C4. Certain chimeric constructs, such as pm-EG and pm-IK (Table V), are not dual-action GAG synthases; they have neither pmHAS nor pmCS activities. However, both pm-EG and pm-IK were not globally misfolded because they retained GlcUA-transferase activity, suggesting rather that the hexosamine transferase domain may be somewhat sensitive to disruption.



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Fig. 4. Schematic model of the two putative glycosyltransferase sites of pmHAS and pmCS. The pmHAS and pmCS enzymes both contain two distinct and relatively independent glycosyltransferase sites. Depicted are the experimentally determined approximate limits of each domain of pmHAS; A1 encompasses residues ~118–427, and A2 encompasses residues ~428–703. Each site possesses a DGS and a DXD amino acid motif. A WGGED motif is found near the junction of the two domains and is involved in hexosamine-transferase activity. The carboxyl terminal residues 704–972 are involved in membrane association (MEM ASSOC) but not required for HAS catalytic activity; deletion of this region converts the enzyme into a soluble form. Residues 1–117 (cross-hatched) are dispensable for catalysis of sugar transfer but may contain structural scaffolding elements or play other roles. PmCS is predicted to use the same structural organization. The region of residues 222–265 of pmHAS or 215–258 of pmCS contains groups important for discriminating between UDP-GlcNAc and UDP-GalNAc.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Potential role of DXD motifs
The DXD motif is conserved in many glycosyltransferases from different families, and the aspartates have been shown to be crucial for activity in enzymes whose function and sequences are highly divergent (Busch et al., 1998Go; Griffiths et al., 1998Go; Wiggins and Munro, 1998Go; Garinot-Schneider et al., 2000Go; Li et al., 2001Go; Keenleyside et al., 2001Go). The pmHAS polypeptide possesses a DXD motif in both Domain A1 and Domain A2. Mutagenesis of any of these four aspartates indicates that they are involved in HA polymerization in agreement with the presumed critical role of the motif. Mutation of Domain A1 DXD results in the loss of GlcNAc-transferase activity, whereas mutation of Domain A2 DXD results in the loss of GlcUA-transferase activity.

Although the importance of the DXD motif was hypothesized, its major function was not clarified until structural evidence was obtained. Based on an X-ray crystal structure of a fragment of SpsA, a putative family 2 glycosyltransferase of currently unknown catalytic activity, the DXD motif is a nucleotide-binding element. The first aspartate forms a hydrogen bond with the ribose ring, and the second aspartate coordinates with the metal cation bound to the phosphate to assist leaving group departure (Charnock and Davies, 1999Go). The involvement of the DXD motif in nucleotide binding and metal ion interaction is supported by several other available glycosyltransferase structures which were solved later, including bovine ß4-galactosytransferase (Gastinel et al., 1999Go), rabbit N-acetylglucosaminyltransferase I (in which the motif is in the form of EDD and the last aspartate, D213, makes the only direct interaction with the bound Mn2+; Ünligil et al., 2000Go), and human ß1,3-glucuronyltansferase I (Pedersen et al., 2000Go). A retaining enzyme, bovine {alpha}1,3-galactosyltransferase, contains a DXD motif with a similar structure for UDP binding (Gastinel et al., 2001Go). However, in contrast to these examples, the GM2 synthase possesses a putative DXD motif that is critical for function, but this motif does not bind the nucleotide directly (Li et al., 2001Go).

In the case of pmHAS—which possesses two separate transferase sites each with a DXD motif—we predict that each transferase site contains a set of UDP precursor binding sites and catalytic residues. Our data suggest that the binding interaction between the two DXD motifs and the UDP-sugar/metal ion substrate complex are not identical. First, the GlcNAc-transferase and the GlcUA-transferase activities of pmHAS differ in their relative preference for Co2+ and Mg2+. The underlying reason for this selectivity is not known, but we speculate that various metal ions confer different coordination angles and geometry to the sugar nucleotide/enzyme binding site. Indeed, the X-ray crystal structure of SpsA showed that the two phosphate groups of UDP are ordered differently in the presence of Mn2+ or Mg2+ (Tarbouriech et al., 2001Go). Second, another difference in the two active sites is that D529E (the second D of the DXD of Domain A1) but not D249E (the comparable residue in Domain A2) possessed low levels of HAS activity.

The difference in the two DXD motifs is also reflected by the performance of mutants in HAS assays in the presence of high amounts (e.g., 20 mM) of the substrate UDP-sugar. The HAS activity of the mutants with glutamate substitutions (the other negatively charged amino acid residue) for both D247 and D249 of Domain A1 could be rescued slightly, but mutants with the neutral residue asparagine, D247N and D249N, were not significantly enhanced at high substrate concentrations. Thus the negative charge of these side chains of this motif element of Domain A1 is important. In experiments probing Domain A2, neither D527E nor D527N mutant could be rescued, but D529E was weakly active in the standard assay and the D529N mutants could also be slightly rescued by high levels of UDP-GlcUA (e.g., 20 mM).

The kinetic studies on the D529E mutant suggest that D529 is involved in precursor binding because the apparent UDP-GlcUA and Mn ion Km values were higher in the mutant than the wild-type enzyme. However, the differences in relative affinity are small, thus further biophysical experiments will be required to resolve this issue.

Putative role of WGGED motif
The WGGED motif was first noted among ß4-galactosyltransferases (Van Die et al., 1997Go), and a similar motif, WGXEXXE, was found among UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (Breton and Imberty, 1999Go). Residues in this Gal/GalNAc-transferase motif have been shown to be essential for enzyme activity (Hagen et al., 1999Go). The X-ray crystal structure of bovine ß4-galactosyltransferase showed that E317D residues in WGGE317D segment are located at the bottom of the proposed UDP-Gal binding pocket (Gastinel et al., 1999Go). They speculated that the E or the D residue was a good candidate for making the nucleophilic attack on the 4-hydroxyl group of the acceptor substrate GlcNAc ring. The assignment of the role of catalytic base to an E or D residue is supported by structural studies on several other glycosyltransferases (Tarbouriech et al., 2001Go).

There is only a single WGGED motif in pmHAS. We found that the GlcNAc-transferase but not the GlcUA-transferase activity of pmHAS depends on the WGGED motif. Boeggeman and Qasba (2002)Go suggested that this sequence element could also be a potential low-affinity metal ion-binding site in bovine ß1,4 galactosyltransferase. However, neither direct structural evidence nor our metal ion rescue experiments support this model for pmHAS. HAS assays performed in the presence of 20 mM UDP-GlcNAc indicate that the HAS activity of E369D could be elevated ninefold in comparison to 0.3 mM UDP-GlcNAc. The activity of E369Q could barely be rescued, indicating that the charge at position 369 is critical. The homologous pmCS enzyme, a CS, also possesses this motif. The WGGED motif may play the same catalytic function in the hexosamine transfer reaction of the Pasteurella synthases as it does in the Gal/GalNAc-transferases, but further biological or structural data are needed for confirmation. The identity of the catalytic base for the GlcUA-transferase active site in Domain A2 is not known at this time; no obvious candidates are apparent based on visually surveying the amino acid sequence of pmHAS.

pmHAS and pmCS: one polypeptide, two transferase sites
Saxena et al. (1995)Go proposed that two types of putative domains, Domain A and Domain B, were present in many ß-glycosyltransferases that use nucleotide diphospho sugars as donors. They noted that processive enzymes, which add a number of sugar residues without releasing the nascent polymer chain, possess both Domains A and B, and those enzymes that add a single sugar residue usually have only Domain A. In general, Domain A resides in the N-terminal half of the polypeptide and possesses two invariant Asp residues, whereas Domain B resides in the C-terminal half and with an invariant Asp residue along with a characteristic QXXRW motif. They hypothesized that the production of heteropolysaccharides with alternating sugar residues, such as HA, is fulfilled by specializing Domain A for one sugar and Domain B for a different sugar.

The only known member of Class II HASs, pmHAS, possesses two tandem copies of Domain A and does not contain Domain B. Our data from the activity analysis of the truncated versions and the point mutants of pmHAS strongly suggest that two active sites coexist in one polypeptide. Overall, pmHAS appears to be a polypeptide with two coordinated but intrinsically nonprocessive activities. Support for this characterization is found in the pmHAS mutant in vitro reconstitution study; two distinct mutant polypeptide molecules can act together to polymerize HA chains in a rapid fashion. The HA chain must be released by one mutant to be acted on by the other mutant in vitro. The distinct Class I HASs, however, do not appear to release the nascent chain during synthesis (DeAngelis and Weigel, 1994Go) and possess both Domain A and Domain B candidates.

The pmCS is 90% identical to pmHAS and possesses two similar sets of putative nucleotide-binding elements. Therefore it is logical that pmCS utilizes the same structural organization and general catalytic mechanism. Dissection of the two transferase activities in pmHAS provides direct evidence for the two active center model (Figure 4). The E. coli K4 chondroitin polymerase (named a polymerase rather than synthase due to its reported absolute requirement for an acceptor sugar), KfoC, was recently published (Ninomiya et al., 2002Go). This protein is about 60% identical to pmHAS and pmCS and thus probably utilizes similar motifs and domains. Another case of the "one polypeptide, two active center" model is the eukaryotic glycosyltransferase FT85, an enzyme involved in the glycosylation of Skp1 protein in Dictyostelium. This bifunctional glycosyltransferase mediates the ordered addition of ß1,3-linked Gal and {alpha}1,2-linked Fuc to the Skp1 glycomoiety. The overall architecture of FT85 resembles pmHAS in that it contains two glycosyltransferase domains (West et al., 2002Go). It is likely that heparosan synthase from Type D P. multocida, pmHS, also utilizes the same strategy (DeAngelis and White, 2002Go). Its N-terminal and C-terminal residues align well with KfiC and KfiA proteins, respectively, which are two of the E. coli K5 proteins required for heparosan biosynthesis (Griffiths et al., 1998Go; Hodson et al., 2001).

In contrast to the Class II pmHAS, the Class I HASs contain both putative Domains A and B. A site-directed mutagenesis study of the mouse HAS1 protein suggested that the conserved amino acid residues in both Domain A (containing a D242XD244) and Domain B (containing Q380XXRW384) were essential for chito-oligosaccharide synthesis (a product made by repeated ß4GlcNAc addition), opposing the hypothesis that the two domains represent two separate activity centers (Yoshida et al., 2000Go). However, in the same report, substitution of Leu314 (in Domain B) caused severe loss of HAS function but not chito-oligosaccharide synthesis, suggesting that in the mouse HAS1 protein, the GlcNAc-transferase activity and the GlcUA-transferase activity can be separated and a two-centers model might be possible. For other processive enzymes, such as bacterial or plant cellulose synthase (forms ß4Glc polymer) and fungal chitin synthase (forms ß4GlcNAc polymer), the polymerization mechanism is also not clear.

UDP-sugar substrate specificity
In recent years, a large number of glycosyltransferases have been identified at the DNA level, but the current knowledge about the enzymes' donor and acceptor specificity is limited to empirical testing and/or identification of their reaction products. Enzymes even within the same family can have a rather broad range of donor and acceptor specificity, making it more difficult to identify the selectivity determinants. There are several X-ray crystal structures for glycosyltransferases, but these static snapshots of catalysis have not provided a clear interpretation of the mechanism of substrate specificity. Although Q289 of E. coli MurG is suggested to play a role in discriminating between UDP-GlcNAc and UDP-GalNAc (Ha et al., 2000Go), this result is limited and the contribution of a single residue might not be sufficient. HA and chondroitin are polysaccharide chains composed of disaccharide repeats that differ at only one sugar; HA contains GlcNAc and chondroitin contains GalNAc, the C-4 epimer of GlcNAc. Domain swapping between pmHAS and pmCS of residues in their N-terminal A1 domains pinpointed a 44-residue region that seems to be involved in differentiating GlcNAc and GalNAc (Figure 4). This region also contains a DXD motif implicated in UDP-sugar binding. Further mutagenesis and/or structural data will be needed to further unravel the selectivity mechanism.

Conclusions
The domain structure and the roles of several putative glycosyltransferase motifs of the Pasteurella HAS were analyzed by characterizing various mutants. Overall, two relatively independent transferase sites exist in one polypeptide. Each transferase site possesses a pair of similar DGS and DXD motifs with essential acidic residues, but the DXD motifs have some distinct biochemical characteristics. Domain-swapping experiments of pmHAS and pmCS indicate that these enzymes utilize the same general mechanism for polymerization. We localized a region of the enzymes near a DXD motif critical for discriminating UDP-GlcNAc from UDP-GalNAc.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Reagents
Molecular biology reagents were from Promega (Madison, WI) unless noted. Custom oligonucleotides were from the Great American Gene Company (Ramona, CA). Oligonucleotides for mutagenesis were trityl purified. All other reagents were the highest grade available from either Sigma (St. Louis, MO) or Fisher Scientific (Silver Spring, MD) unless otherwise noted.

Generation of pmHAS truncations and site-directed mutagenesis
Truncated polypeptides were generated by amplifying the pPm7A insert (DeAngelis et al., 1998Go) by 13 cycles of PCR with Taq DNA polymerase (Fisher) and synthetic oligonucleotide primers corresponding to various portions of the pmHAS open reading frame. To generate pmHAS46–160/428–703, pmHAS46–160 and pmHAS428–703 were first amplified. The reverse primer for pmHAS46–160 contained overlap with the forward primer for pmHAS428–703. The two purified PCR products and the outer pair of PCR primers were then used for another round of PCR. Except for the construction of pmHAS1–686 and pmHAS1–668, the PCR primers contained EcoRI and PstI restriction sites to facilitate cloning into the expression plasmid pKK223-3 (tac promoter; Pharmacia, Uppsala, Sweden). The resulting recombinant constructs were transformed into E. coli TOP10F' cells (Invitrogen, Carlsbad, CA) and maintained at 30°C on Luria-Bertani media with ampicillin selection. The DNA encoding pmHAS1–686 and pmHAS1–668 were cloned into pETBlue-1 plasmid and expressed in E. coli Tuner(DE3)pLacI cells (Novage, Madison, WI) according to the manufacturer's instructions; these cells were maintained at 30°C on Luria-Bertani media with carbenicillin and chloramphenicol selection.

Constructs with point mutations were generated using the QuickChange site-directed mutagenesis method (Stratagene, La Jolla, CA) 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 preparations
Recombinant E. coli were grown in Luria-Bertani media at 30°C with drug selection until the culture's A600 reached 0.3–0.6 when cells were induced with 0.5 mM isopropyl-1-thio-ß-D-galactoside. Cells were harvested 5 h after induction. For soluble truncated proteins and pmHAS1–703-derived mutants expressed in E. coli TOP10F' cell, cells were extracted with B-Per II Bacterial Protein Extraction Reagent (an octylthioglucoside-based solution; Pierce, Rockford, IL) according to the manufacturer's instruction except that the procedure was performed at 7°C in the presence of a protease inhibitor cocktail. For proteins expressed in E. coli Tuner(DE3)pLacI, lysis by ultrasonication followed by subcellular fractionation was performed (Jing and DeAngelis, 2000Go) and the supernatant after centrifugation at 100,000 x g was used.

Various enzyme assays for polysaccharide polymerization or for single-sugar transfer
Five assays were designed to detect either (1) the polymerization of long HA chains, (2) the addition of a single GlcNAc to a GlcUA-terminated HA oligosaccharide acceptor, (3) the addition of a single GlcUA to a GlcNAc-terminated HA oligosaccharide acceptor, (4) the polymerization of long chondroitin chains, or (5) the addition of a single GalNAc to a GlcUA-terminated HA oligosaccharide acceptor. For the typical reaction, conditions using normal levels of UDP-sugars (0.15 mM UDP-GlcUA, 0.3 mM of UDP-GlcNAc; 25 µl reaction), the first three assays were described previously (Jing and DeAngelis, 2000Go). In certain cases as noted, higher level of UDP-sugars (up to 20 mM) were used in an attempt to compensate for the mutant HASs' poor activity. For the CS assay, the same conditions as the normal HAS assay were used except that the other hexosamine precursor, UDP-GalNAc, was employed and there is no ammonium sulfate or ethylene glycol in the assay system.

GalNAc-transferase activity was assayed under the same conditions as the GlcNAc-transferase assay except that 0.3 mM UDP-[3H]GalNAc (0.2 µCi) (NEN, Boston, MA) was used instead of UDP-[3H]GlcNAc. Reactions were terminated by the addition of sodium dodecyl sulfate to 2% (w/v). The reaction products were separated from unincorporated substrates by descending paper (Whatman 3 M) chromatography with ethanol/1 M ammonium acetate, pH 5.5, development solvent (65:35 for the HAS, CS, and GlcUA-transferase assays; 75:25 for GlcNAc-transferase and GalNAc-transferase assay). All assays were performed in duplicate and adjusted to be linear with regard to incubation time and to protein concentration. Radiolabeled products were quantitated by liquid scintillation counting (Biosafe II; Research Products International, Mt. Prospect, IL).

Apparent Km determination
HAS assays (25 µl) were carried out in duplicate with a fixed saturating amount of UDP-[3H]GlcNAc (1 mM, 0.1 µCi) and HA acceptor (0.12 mg/ml) but increasing amounts of UDP-GlcUA (0–1.2 mM) for determining the UDP-GlcUA substrate apparent Km. For the metal ion apparent Km, fixed amounts of UDP-sugars (1 mM UDP-GlcNAc and 1 mM UDP-[14C]GlcUA, 0.04 µCi) and increasing amounts of Mn2+ (0–1.5 mM) were used. The reactions were incubated at 30°C for 45 min. The paper chromatography method was then performed, and radioactivity at the origin corresponding to HA polymer was measured.

Western blot analysis
The pmHAS, pmHAS mutants, pmCS, and chimeric polypeptides in extracts were separated using standard 8% polyacrylamide sodium dodecyl sulfate gels and detected by western blotting using a monospecific antibody directed against a synthetic peptide corresponding to residues 526–543 of pmHAS (acetyl-LDSDDYLEPDAVELCLKE-amide) as described in Jing and DeAngelis (2000)Go. This reagent is cross-reactive with pmCS because the enzyme contains the identical sequence element. To ensure that an equivalent amount of each enzyme was tested in assays, a titration of wild-type pmHAS extract and each of the pmHAS mutant extracts were directly compared by western blot analysis.

Domain swapping constructs
The DNA encoding different segments of pmHAS or pmCS were generated by amplifying the pPm7A insert (DeAngelis et al., 1998Go) or pPmF4A insert (DeAngelis and Padgett-McCue, 2000Go), respectively, by 15 cycles of PCR with Taq DNA polymerase (Fisher) and synthetic oligonucleotide primers corresponding to various portions of the pmHAS or pmCS open reading frame. Each internal primer contained overlaps with the other segment to allow joining of the two desired segments. The forward and reverse primers for pmHAS residue 1–427 (A segment) were P1 = 5'-ATGAACACATTATCACAAGCAATAAAAGC-3' and P2 = 5'-GCGAATCTTCTATTGGTAAAAGYTTTC-3' (Y = C/T), respectively. The forward and reverse primers for pmCS residue 421–704 (C segment) were P3 = 5'-CTTTTACCAATAGAAGATTCGCATAT-3' and P4 = 5'-GAAGACGTCTTAGGCATCTTTATTCTGAATGAG-3', respectively. The forward and reverse primers for pmCS residue 1–420 (D segment) were P1 and P2. The forward and reverse primers for pmHAS residue 428–703 (B segment) were P3 and P5 = 5'-GGGAATTCTGCAGTTAAATATCTTTTAAGATATCAATCTCTTC-3', respectively. The forward and reverse primer for pmHAS residue 1–265 (E segment) were P1 and P6 = 5'-AACAATATCATTGTCTTCTAATAGCTCTGCAACATAAG-3'. The forward and reverse primer for pmCS residue 259–704 (G segment) were P7 = 5'-GAAGACAATGATATTGTTTTAATTGG-3' and P4. The forward and reverse primer for pmHAS residue 266–703 (F segment) were P8 = 5'-GAAGATGATGATTTAACAATCATTGG-3' and P5. The forward and reverse primer for pmCS residue 1–258 (H segment) were P1 and P9 = 5'-GTTAAATCATCATCTTCTAATAGTTCTGTAAGATAAG-3'. The forward and reverse primer for pmHAS residue 1–221 (I segment) were P1 and P10 = 5'-CAATTGATATCCATAATCTTTTTGTCTGACGTAGCG-3'. The forward and reverse primer for pmCS residue 215–704 (K segment) were P11 = 5'-GATTATGGATATCAATTGTGTGCAG-3' and P4. The forward and reverse primer for pmHAS residue 222–703 (J segment) were P12 = 5'-GATAACGGTTTTCAAGCCAGTGCCG-3' and P5. The forward and reverse primer for pmCS residue 1–214 (L segment) were P1 and P13 = 5'-GGCTTGAAAACCGTTATCTTTTTGTCTTACATACTTTATG-3'.

The chimeric synthases were created by 15 cycles of PCR with the agarose gel–purified (GeneClean; Bio101, Vista, CA) segments and outer primers (pm-AC used A and C segments with primers P1 and P4; pm-BD used B and D segments with primers P1 and P5; pm-EG used E and G segments with primers P1 and P4; pm-FH used F and H segments with primers P1 and P5; pm-IK used I and K segments with primers P1 and P4; pm-JL used J and L segments with primers P1 and P5). The purified PCR products were cloned into pETBlue-1 vector and the chimeric proteins were expressed in E. coli Tuner(DE3)pLacI cells (Novagen). We sequenced the complete open reading frames of multiple clones of both constructs. We found a pm-AC construct that was perfect, but both of the two pm-BD constructs that we had sequenced completely had secondary undesired mutations (#1, E695 and I697F; #2, I302V). However, these mutations were in different locations and the enzymes' transferase activities were identical. Several other pm-BD clones have the identical enzyme phenotypes, but their complete sequences were not determined.


    Acknowledgements
 
This work was supported by grants from the National Science Foundation (MCB-9876193) and the National Institutes of Health (R01-GM56497) to P.L.D. We thank Drs. Ron Bowditch, Richard Cummings, Rodney Tweten, Jordan Tang, and Paul Weigel for comments concerning these studies.


    Footnotes
 
1 Present address: Hyalose LLC, 655 Research Parkway, Suite 525, Oklahoma City, OK 73104. Back

2 To whom correspondence should be addressed; email: paul-deangelis{at}ouhsc.edu Back


    Abbreviations
 
CS, chondroitin synthase; GAG, glycosaminoglycan; HA, hyaluronan; HAS, hyaluronan synthase; PCR, polymerase chain reaction


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 Top
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 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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