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
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Abstract |
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Key words: capsule / chondroitin / glycosaminoglycan / glycosyltransferase / hyaluronan
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Introduction |
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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, 2002). 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., 1998). 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, 1999b
). 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, 2000
). Within the pmHAS sequence, there is a pair of duplicated domains that are similar to the Domain A proposed by Saxena et al. (1995)
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., 2002), 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, 2000
). The pmCS contains 965 amino acid residues and is about 90% identical to pmHAS. A soluble recombinant Escherichia coliderived pmCS1704 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.
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Results |
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Site-directed mutagenesis of pmHAS1703 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., 1995). pmHAS has two Domain Alike regions; we therefore called the regions Domains A1 and A2 (Jing and DeAngelis, 2000
) (Figure 1). According to the hydrophobic cluster analysis program (B. Henrissat personal communication), Domains A1 and A2 encompass residues 152325 and 432604, 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, 2000
). We also noted the existence of a third potential DGS sequence motif in pmHAS located at position 563565. To determine if this motif was as critical for synthase activity, like the other two DGS motifs, we mutated D563 of pmHAS1703 into a glutamate, asparagine, or lysine residue. All of these mutants behaved like wild-type pmHAS1703 (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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Site-directed mutagenesis of pmHAS1703 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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In vitro reconstitution of HAS activity with two distinct mutant proteins
Previously we demonstrated that a combination of two pmHAS1703 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, 2000). 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 (410 sugars long), all of the combinations of enzymes synthesized HA polymer. In most cases, the relative polymerization efficiencies were close to wild-type pmHAS1703 (
30100%). 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., 2003
). 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, 2000). 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., 1989
). We used the active truncated versions of the synthases, pmCS1704 and pmHAS1703, 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 1427 from pmHAS and residues 421704 from pmCS (pm-AC construct) was an active HAS. The opposite combination, an enzyme consisting of residues 1420 from pmCS and residues 428703 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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Discussion |
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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, 1999). 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., 1999
), 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., 2000
), and human ß1,3-glucuronyltansferase I (Pedersen et al., 2000
). A retaining enzyme, bovine
1,3-galactosyltransferase, contains a DXD motif with a similar structure for UDP binding (Gastinel et al., 2001
). 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., 2001
).
In the case of pmHASwhich possesses two separate transferase sites each with a DXD motifwe 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., 2001). 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., 1997), and a similar motif, WGXEXXE, was found among UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (Breton and Imberty, 1999
). Residues in this Gal/GalNAc-transferase motif have been shown to be essential for enzyme activity (Hagen et al., 1999
). 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., 1999
). 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., 2001
).
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) 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) 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, 1994) 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., 2002). 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
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., 2002
). It is likely that heparosan synthase from Type D P. multocida, pmHS, also utilizes the same strategy (DeAngelis and White, 2002
). 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., 1998
; 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., 2000). 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., 2000), 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.
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Materials and methods |
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Generation of pmHAS truncations and site-directed mutagenesis
Truncated polypeptides were generated by amplifying the pPm7A insert (DeAngelis et al., 1998) 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 pmHAS46160/428703, pmHAS46160 and pmHAS428703 were first amplified. The reverse primer for pmHAS46160 contained overlap with the forward primer for pmHAS428703. 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 pmHAS1686 and pmHAS1668, 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 pmHAS1686 and pmHAS1668 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/pmHAS1703 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 0.30.6 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 pmHAS1703-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, 2000) 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, 2000). 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 (01.2 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+ (01.5 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 526543 of pmHAS (acetyl-LDSDDYLEPDAVELCLKE-amide) as described in Jing and DeAngelis (2000). 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., 1998) or pPmF4A insert (DeAngelis and Padgett-McCue, 2000
), 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 1427 (A segment) were P1 = 5'-ATGAACACATTATCACAAGCAATAAAAGC-3' and P2 = 5'-GCGAATCTTCTATTGGTAAAAGYTTTC-3' (Y = C/T), respectively. The forward and reverse primers for pmCS residue 421704 (C segment) were P3 = 5'-CTTTTACCAATAGAAGATTCGCATAT-3' and P4 = 5'-GAAGACGTCTTAGGCATCTTTATTCTGAATGAG-3', respectively. The forward and reverse primers for pmCS residue 1420 (D segment) were P1 and P2. The forward and reverse primers for pmHAS residue 428703 (B segment) were P3 and P5 = 5'-GGGAATTCTGCAGTTAAATATCTTTTAAGATATCAATCTCTTC-3', respectively. The forward and reverse primer for pmHAS residue 1265 (E segment) were P1 and P6 = 5'-AACAATATCATTGTCTTCTAATAGCTCTGCAACATAAG-3'. The forward and reverse primer for pmCS residue 259704 (G segment) were P7 = 5'-GAAGACAATGATATTGTTTTAATTGG-3' and P4. The forward and reverse primer for pmHAS residue 266703 (F segment) were P8 = 5'-GAAGATGATGATTTAACAATCATTGG-3' and P5. The forward and reverse primer for pmCS residue 1258 (H segment) were P1 and P9 = 5'-GTTAAATCATCATCTTCTAATAGTTCTGTAAGATAAG-3'. The forward and reverse primer for pmHAS residue 1221 (I segment) were P1 and P10 = 5'-CAATTGATATCCATAATCTTTTTGTCTGACGTAGCG-3'. The forward and reverse primer for pmCS residue 215704 (K segment) were P11 = 5'-GATTATGGATATCAATTGTGTGCAG-3' and P4. The forward and reverse primer for pmHAS residue 222703 (J segment) were P12 = 5'-GATAACGGTTTTCAAGCCAGTGCCG-3' and P5. The forward and reverse primer for pmCS residue 1214 (L segment) were P1 and P13 = 5'-GGCTTGAAAACCGTTATCTTTTTGTCTTACATACTTTATG-3'.
The chimeric synthases were created by 15 cycles of PCR with the agarose gelpurified (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.
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; email: paul-deangelis{at}ouhsc.edu
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Abbreviations |
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References |
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