Acceptor Specificity of the Pasteurella Hyaluronan and Chondroitin Synthases and Production of Chimeric Glycosaminoglycans*

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (∼102-4 sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.

these glycosyltransferase activities that apparently interacts with three or four saccharide units of the nascent HA chain (18). The sugar nucleotide specificity of native sequence enzymes has been evaluated with naturally occurring UDPsugar donors; only the authentic monosaccharide molecules are transferred efficiently by the native enzymes (e.g. C-4 epimers will not substitute). All known Pasteurella enzymes add single sugars in a repetitive stepwise fashion to the nonreducing terminus of their cognate acceptor oligosaccharides (13, 17,19). Here we report that PmHAS and PmCS will elongate a range of acceptor molecules in addition to their cognate sugars; this finding sheds light on the nature of the synthase active sites as well as significantly expanding the potential repertoire of oligosaccharide and polysaccharide targets that may now be synthesized.

EXPERIMENTAL PROCEDURES
Natural GAGs-All GAG polysaccharides, except for HA, are heterogeneous in nature with respect to composition and sulfation pattern. It is currently impossible to isolate GAG polysaccharides from animal sources in one pure isomeric form. Therefore, throughout the text all polysaccharides are defined with respect to their source and are given a simple nomenclature code (Table 1).
A variety of GAGs were obtained from Sigma, including Streptococcus zooepidemicus hyaluronan (HA), bovine trachea chondroitin sulfate (CS bt ) (sold as "chondroitin sulfate A"; mainly composed of chondroitin 4-sulfate), C-5 epimerized porcine intestinal mucosa chondroitin sulfate (CS pI ) (also referred to as dermatan sulfate and sold as "chondroitin sulfate B"), shark cartilage chondroitin sulfate (CS s ) (sold as "chondroitin sulfate C"; mainly composed of chondroitin 6-sulfate), and porcine intestinal heparin (Hep). Shark fin chondroitin sulfate (CS sf ) (sold as "chondroitin sulfate D") and squid cartilage chondroitin sulfate (CS q ) (sold as "chondroitin sulfate E") were obtained from Associates of Cape Cod/Seikagaku America (Falmouth, MA). In addition, a highly C-5 epimerized (Ͼ95% 4S-GalNAc-IdoUA repeats) chondroitin sulfate (CS pII ; a high purity dermatan sulfate from porcine intestinal mucosa similar to CS pI above, but better characterized) and heparan sulfate (porcine intestinal mucosa) were obtained from Celsus Laboratories (Cincinnati, OH). Unsulfated chondroitin polysaccharide (C) and unsulfated heparosan polysaccharide was prepared from cultures of either type F or type D P. multocida, respectively (20). The degree of polymerization of the HA and the unsulfated chondroitin was reduced to ϳ80 and ϳ20 kDa, respectively, by autohydrolysis (121°C, 20 p.s.i., 20 min) to be more comparable with the smaller molecular weight chondroitin sulfates (ϳ15-40 kDa) and heparin (ϳ17-19 kDa). In addition, 80-kDa monodisperse HA was prepared by synchronized stoichiometrically controlled chemoenzymatic reactions (12).
To avoid the heterogeneity problem intrinsic to natural GAG polysaccharides, a series of defined oligosaccharides of known  Table 2.
Chondroitin 4-Sulfate Trisaccharide (CS bt3 )-Chondroitin 4-sulfate from bovine trachea, CS bt (1.0 g), was treated with chondroitin ABC lyase (EC 4.2.2.4, Proteus vulgaris; Sigma; 1 unit) in 50 mM Tris-HCl/sodium acetate buffer at 37°C to obtain a mixture of unsaturated oligosaccharides. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 (Bio-Rad) column eluted with 100 mM sodium chloride. Sized oligosaccharides were then desalted by SEC on a Bio-Gel P2 column. Charge separation of the tetrasaccharide fraction was carried out by semi-preparative strong anion exchange-high performance liquid chromatography (SAX-HPLC) (21). The major peaks were pooled, lyophilized, and desalted on a Bio-Gel P2 column. The unsaturated chondroitin sulfate tetrasaccharide containing two 4-O-sulfonated GlcNAc residues (1 mg) was dissolved in distilled water (1 mg/ml). To remove the nonreducing terminal unsaturated GlcUA residue, equal volumes of the oligosaccharide solution and mercuric acetate reagent (35 mM, adjusted to pH 5 using acetic acid in distilled water) were added and stirred for 15 min at room temperature. The reaction mixture was then passed through a pre-washed Dowex 50W-X8 H ϩ column, neutralized with saturated sodium bicarbonate solution, and then applied to a Bio-Gel P2 column to obtain the pure trisaccharide CS b3 . The structure of this trisaccharide was confirmed by ESI-MS analysis (Table 3) (21).
Chondroitin 6-Sulfate Trisaccharide (CS s3 ) and Pentasaccharide (CS s5 )-Chondroitin 6-sulfate from shark cartilage, CS s (50 mg), was treated with chondroitin ABC lyase (0.05 units) as above. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 column, and each oligosaccharide was desalted on a Bio-Gel P2 column. The unsaturated chondroitin tetrasaccharide (1 mg) and hexasaccharide (1 mg) were treated with mercuric acetate reagent as described earlier to obtain the trisaccharide CS s3 and pentasaccharide CS s5 . The structures of these products were determined by ESI-MS analysis ( Table 3).
C-5-epimerized Chondroitin 4-Sulfate Trisaccharide and Pentasaccharide (CS pII3 and CS pII5 )-C-5-epimerized chondroitin sulfate from porcine intestinal mucosa, CS pII (Celsus; 10 g), was treated with chondroitin ABC lyase (20 units) as above. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 column, and each oligosaccharide was desalted by SEC on a Bio-Gel P2 column. Charge separation of sized oligosaccharide fractions was carried out by SAX-HPLC. The major peaks were pooled, lyophilized, and desalted on a Bio-Gel P2 column. The unsaturated C-5-epimerized chondroitin sulfate tetrasaccharide (6 mg) and hexasaccharide (2 mg) were treated with mercuric acetate reagent to obtain the pure C-5-epimerized chondroitin sulfate trisaccharide, CS pII3 , and pentasaccharide, CS pII5 . The structures of these oligosaccharides were confirmed by 1 H NMR and ESI-MS analysis ( Table 3) (21).
Desulfated C-5-epimerized Chondroitin Sulfate (dCS pII ) Polysaccharide-The sodium salt of C-5-epimerized chondroitin sulfate (high purity dermatan sulfate from porcine intestinal mucosa; Celsus), CS pII (250 mg), was treated with 250 ml of acidic methanol (1.25 ml of acetyl chloride in 250 ml of methanol) for 3 days at room temperature. The recovered solid product (methyl ester of desulfated epimerized chondroitin sulfate) was treated with 10 ml of 0.1 M sodium hydroxide for 24 h at room temperature to obtain free carboxylate, O-desulfated epimerized chondroitin sulfate dCS pII (22). This structure was confirmed by 1 H NMR spectroscopy (23).
Desulfated C-5-epimerized Chondroitin Sulfate Trisaccharide (dCS pII3 )-The dCS pII polymer (100 mg) was treated with chondroitin ABC lyase (1 unit) as described earlier. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 column, and each oligosaccharide was desalted on a Bio-Gel P2 column. Charge separation of sized oligosaccharide fractions was carried out by SAX-HPLC. The major peaks were pooled,  Synthase Acceptor Specificity JANUARY 5, 2007 • VOLUME 282 • NUMBER 1 lyophilized, and desalted on a Bio-Gel P2 column. C-5-epimerized chondroitin tetrasaccharide without sulfate groups at Glc-NAc residues (1 mg) was treated with mercuric acetate reagent to obtain the trisaccharide. The structure of this trisaccharide was confirmed by ESI-MS analysis (Table 3). Synthetic Heparin Pentasaccharide (Hep 5 )-The synthetic analog of the anti-thrombin III binding pentasaccharide, fondaparinux sodium (Arixtra), was obtained from the pharmacy. The contents of 10 syringes (25 mg in 5 ml of isotonic saline total) were collected and dialyzed (1000-Da cutoff) against water for 2 days at 4°C and lyophilized to obtain the pentasaccharide Hep 5 in quantitative yield.
Heparosan Pentasaccharide (H e5 )-Heparosan polysaccharide was isolated from E. coli K5 and purified according to the published procedure (24). Using heparin lyase III from Flavobacterium heparinum, a controlled partial depolymerization was performed. The oligosaccharide mixture was size-fractionated using a Bio-Gel P6 column. Each fraction was concentrated and desalted on a Bio-Gel P2 SEC column. The unsaturated hexasaccharide was treated with mercuric acetate reagent to obtain heparosan pentasaccharide H e5 . Analysis by ESI-MS confirmed the structure of this product (data not shown).
Sugar Quantitation-All carbohydrates were assayed by carbazole method (25) for uronic acid using GlcUA as a standard.
Synthase Purification-The recombinant E. coli cells expressing the truncated, soluble dual-action catalysts PmHAS-(1-703) or PmCS-(46 -695) were extracted with the permeabilization agent octylthioglucoside (1% w/v) in 1 M ethylene glycol, 50 mM Tris, pH 7.2, containing protease inhibitors (14). The extract was clarified by centrifugation and applied to a pseudoaffinity column (Tosoh Toyopearl AF-Red-650) equilibrated in the same buffer except 50 mM Hepes was substituted for Tris. The protein eluted with a NaCl gradient (0 -1.5 M NaCl for 120 min), and the enzyme peak was determined by Coomassie Blue staining of SDS-polyacrylamide gels. The pooled enzyme (ϳ95% pure PmHAS or PmCS) was concentrated with an ultrafiltration device (Amicon Ultra 15 ml, 50-kDa cutoff). The protein was quantitated by the Bradford assay with a bovine serum albumin standard (Pierce).
Synthase Assays and Acceptor Utilization Tests-PmHAS or PmCS-catalyzed polymerization was measured with radiolabeled sugar incorporation assays. UDP-GlcUA and UDP-Hex-NAc polymerization was recorded by monitoring incorporation of UDP-[ 3 H]GlcUA as denoted in Equation 1, acceptor ϩ n UDP-͓ 3 H͔GlcUA ϩ n UDP-HexNAc 3 2n UDP ϩ ͓͑ 3 H͔GlcUA-HexNAc͒ n Ϫ acceptor (Eq. 1) Typically, 25-l reactions containing ϳ1 g (ϳ13 pmol) of enzyme, PmHAS-(1-703) or PmCS-(46 -695), and ϳ0.01 nmol to 3.75 nmol of a given acceptor were employed. A "no acceptor" control was used for all analyses; this background value corresponding to de novo polymer initiation was subtracted from the value obtained from the assay with acceptor. Assays with an HA polymer acceptor were employed in every data set to allow for normalization. Polymerization reactions contained 50 mM Tris, pH 7.2, 1 M ethylene glycol, 2 mM MnCl 2 , 0.05 mM UDP-[ 3 H]GlcUA (1 Ci; PerkinElmer Life Sciences), and 0.2 mM UDP-HexNAc (UDP-GlcNAc for PmHAS or UDP-Gal-NAc for PmCS) and were incubated at 30°C for either 3 min (polysaccharide acceptors) or 30 min (oligosaccharide acceptors). Reactions were stopped by placing on ice and adding SDS (2% final w/v). Descending paper chromatography was used to separate the unincorporated radiolabel from the elongated acceptors (10). The assays were performed in duplicate and were linear with respect to enzyme concentration and time; less than 5% UDP-sugar substrate was consumed.
Single sugar additions were also monitored by reverse phase HPLC ESI-MS (26). Briefly, similar reaction conditions were utilized as described above, except only single unlabeled UDPsugar (2-6 mM final) was employed. The enzyme adds a monosaccharide unit onto the nonreducing end of various acceptors as shown in Equation 2 or 3.

RESULTS AND DISCUSSION
The finding that recombinant PmHAS, and later PmCS, was able to elongate certain small oligosaccharides, including HA 4 and chondroitin sulfate hexasaccharides, respectively (13, 19), led to a broader investigation of the potential range of acceptor usage (Tables 4 and 5). In contrast, the other distinct bacterial HA synthase enzymes from group A and C Streptococcus cannot be tested at this time because of their inability to elongate exogenously added acceptors.
E. coli KfoC, chondroitin polymerase, was shown previously to elongate certain polysaccharides and oligosaccharides, but the polymers were not rigorously defined with respect to sulfation isomers (15). Also, the desulfated chondroitin preparations may have suffered oxidative degradation that hindered enzy-matic utilization (15). Our data show that both HA and unsulfated chondroitin polysaccharides are very good acceptors for HA or chondroitin polymerization by PmHAS or PmCS, respectively ( Table 4). The difference between the HA and the chondroitin backbone is the identity of the hexosamine residue (Fig. 1). HA contains GlcNAc while chondroitin contains the C-4 epimer, GalNAc. For both PmHAS and PmCS, it appears that the acceptor site or nascent chain retention site of the enzyme probably does not recognize the C-4 hydroxyl group by a direct bonding interaction or does not form a close association with this region of the pyranose ring. As mentioned earlier, KfoC was reported to elongate chondroitin worse than chondroitin sulfate, but as those authors admitted, the solvolytic desulfation reaction may have damaged the polymer. On the other hand, the Pasteurella chondroitin tested here is an unsulfated polymer thus eliminating any issues of potential damage because of side reactions during solvolysis. Both PmHAS and PmCS proteins also utilize the small HA tetrasaccharide, HA 4 , with high efficiency ( Table 5).
The conundrum of an impure bulk GAG polymer population found in natural extracts was addressed here by employing defined (i.e. HPLC-purified, mass spectrometry, and NMR-validated) oligosaccharides with known sulfation patterns. The nature of the sulfation pattern seems to be an important characteristic on the extent of utilization of the modified acceptor by both PmCS and PmHAS. For example, the 6-sulfated chondroitin trisaccharide (CS s3 ) and chondroitin pentasaccharide (CS s5 ) served as relatively good acceptors for PmHAS. In contrast, the 4-sulfated chondroitin trisaccharide (CS bt3 ) preparation was a poor acceptor (Table 5). In fact, the signals in the radiolabeled incorporation assay of CS bt3 appear to be due entirely to a trace amount of pentasaccharide with mixed 4-and 6-sulfates contaminating the preparation based on ESI-MS of the PmHAS-extended mixture. The starting CS bt3 trisaccharide peak (calculated 760.12 Da; observed 759.1) was not extended into the predicted tetrasaccharide product (calculated 936.15 Da); a major fraction of the pentasaccharide, however, was extended into a hexasaccharide possessing the appropriate mass. The bulk 4-sulfated chondroitin polysaccharide (CS bt ) did serve as an acceptor for PmHAS and PmCS, but this polymer is not composed entirely of the 4-sulfated isomer; these preparations are actually a 70:30 mixture of GalNAc-4/6SO 4 isoforms ( Table 1). The 6-sulfated sequences may be responsible for the signal observed. In summary, as long as the nonreducing terminal Gal-NAc residue either possesses a 6-sulfate group or lacks a sulfate group, both Pasteurella enzymes should elongate the polymer quite readily. These findings suggest that a bulky negative group at the C-4 position of the hexosamine is causing an unfavorable interaction (steric and/or charge repulsion) with the synthase polypeptide because the HA and unsulfated chondroitin are good acceptors. The 6-sulfate trisaccharide is better tolerated suggesting that the C-6 hydroxyl of the HexNAc (Fig. 1) is neither in close proximity nor in a bonding interaction with the synthase.
Two other sulfated chondroitin polysaccharides containing GalNAc-6SO 4 , CS sf and CS q , show some activity as acceptors (Table 4), but both CS sf and CS q also have an additional sulfate group; CS sf contains GlcUA-2SO 4 , whereas CS q contains Gal-NAc-6/4-disulfate. These polymers serve as modest acceptors (2.4 and 5.1%, respectively, for PmHAS) but are not as good as CS bt (12%) and CS s (10%). Again, the relative activity of the various natural chondroitin preparations to HA may be affected by the fact that these polymers are not 100% pure and can contain additional minor sequences at the nonreducing termini.

TABLE 4 PmHAS and PmCS polysaccharide acceptor specificity
Polymerization assays with each acceptor (at least three experiments in duplicate) were performed. The value of no acceptor control (ϳ300 -600 dpm) was subtracted from each point. The averaged values for PmHAS or PmCS are presented. The ratio (nanomoles of test acceptor/nmol of HA standard) was employed to normalize to the HA signal (30,000 dpm for 0.02 nmol of HA), which was set to 100%. For all results, * ϭ p Ͻ 0.05 and ** ϭ p Ͻ 0.01 in a Student's one-tailed t test for comparison of a given test acceptor to the no acceptor control assay. ND indicates not determined. Both enzymes may utilize either HA or chondroitin polymers, but heparin and heparosan are very poor acceptors or slightly inhibitory.
Desulfated C-5-epimerized chondroitin sulfate B (dCS pII ) was also assayed to determine whether the IdoUA or sulfate groups (Fig. 1) were the problematic structures for the synthases. The desulfated C-5-epimerized polysaccharide, dCS pII , showed 2.4% activity relative to HA, higher than that of the 4-sulfated epimerized chondroitin polymers (CS pI and CS pII , 0.4 and 0.5%, respectively; see Table 4) but lower than that of the unsulfated C polymer (54%). We hypothesized that PmHAS and PmCS may be able to tolerate IdoUA to some degree due to the increase in signal in dCS pII (after de-sulfation) when compared with CS pI and CS pII . There is a chance, however, that IdoUA was not well tolerated. The observed signal may have actually been because of the nonreducing terminus of some chains in the polysaccharide population containing an accessible GlcUA, the native uronic acid for the synthase, instead of IdoUA (i.e. CS pII , the original material that was de-sulfated to make dSC pII , contains ϳ95% IdoUA, and thus there is an ϳ5% chance of the GlcUA residing at the nonreducing terminus of the polymers). To further address this issue, a defined IdoUAcontaining trisaccharide, dCS pII3 , was subjected to a single sugar addition reaction (Eq. 2) with PmHAS. Reverse phase HPLC-ESI-MS analysis verified that the trisaccharide (calcu-lated 600.2 Da; observed 599.2 Da) was converted to the expected tetrasaccharide (calculated 776.23 Da; observed 775.1 Da). Therefore, IdoUA is tolerated by PmHAS. An exact comparison of the GlcUA versus IdoUA utilization rates will need to await the preparation of larger amounts of defined dCS pII3 oligosaccharide. The difference between the GlcUA and the IdoUA epimers is the conformation of the sugar ring. Even though both monosaccharide units have the carboxylate in the equatorial position, two different chair forms ( 4 C 1 versus 1 C 4 for GlcUA or IdoUA, respectively) are present and changes the linkage from ␤ to ␣ upon epimerization.
Two antithrombotics, the synthetic heparin pentasaccharide, Arixtra (Hep 5 ), and bulk heparin polysaccharide (Hep), show very low (0.03%, Hep) relative acceptor activity to HA or were inhibitory (Ϫ0.1%, Hep 5 ) with PmHAS (Tables 4 and 5). The unsulfated heparosan oligosaccharide (1.6%, H e5 ) and polysaccharide (0.1%, H) tested show slightly more activity than the sulfated heparin oligosaccharide and polysaccharide, respectively. It may be that the repeated GlcUA groups among all acidic GAGs can partially compensate for the improper sugar backbone linkages, albeit poorly. However, too many additional negative groups (i.e. sulfate groups) may also be detrimental.
Overall, PmCS exhibited similar acceptor specificity relative to PmHAS (Tables 4 and 5) except that the various chondroitin oligosaccharides were better acceptors for the chondroitin synthase, perhaps reflecting its optimization for chondroitin biosynthesis. A few minor potential differences among the various acceptors were noted, but at this time limiting amounts of sugar reagents precluded further ranking.
Perhaps it is unexpected that the PmHAS and PmCS enzymes would elongate noncognate or sulfated GAGs, but it is not illogical. Some glycosyltransferases have been reported to be promiscuous with respect to their substrate recognition. Also, in living bacteria, the synthase enzymes in the cell interior are not expected to be confronted with noncognate acceptor molecules; thus these catalysts do not need to be selective. On the other hand, the synthase donor sites (especially the UDPhexosamine pocket) are exposed to structurally similar UDPsugars, and thus more stringent selectivity for the authentic substrate is expected and is observed.
A potential biotechnological utility of the relaxed acceptor usage (i.e. the ability of these particular synthases/polymerases to accept unnatural substrates) is the creation of GAG molecules with two or more types of sugar repeating units. A variety of chimeric polymers consisting of two distinct GAG polysaccharides fused together were produced (Figs. 2 and 3). The synthase-catalyzed reactions were rapid (complete within 2-6 h). The length of the added HA or chondroitin chain may be con- trolled by altering the stoichiometry of UDP-sugar to acceptor (12). For example, chondroitin sulfate was extended by PmHAS with HA chains ranging from ϳ400 kDa (ϳ2,000 saccharides; Fig. 3) to ϳ1,600 kDa (ϳ8,000 saccharides) depending on the donor/GAG acceptor ratio. In addition, the chimeric polymers appear to be relatively monodisperse populations (for example, in Fig. 3, polydispersity Mw/Mn values: HA-CS bt , 1.006 Ϯ 0.02; C-HA, 1.003 Ϯ 0.02; for reference, "1" is the ideal polymer preparation).
We verified that HA was not contaminating the chondroitin sulfate preparations by pre-treating with HA lyase; this enzyme produces HA oligosaccharides with defective unsaturated nonreducing ends that cannot be extended by Pasteurella synthases (data not shown). In addition, the Stains-All detection method for agarose gels can distinguish chondroitin sulfate from HA. The sulfated polymers yield a purple or yellow color (depending on sulfation level), whereas the HA is blue; the chimeric molecules have a purple color readily distinguishable from the HA alone (supplemental data, Fig. S1). Furthermore, the chimeric nature of the new GAGs is demonstrated by treatment with specific HA lyase (Fig. 3). For example, an HA chain was extended with a chondroitin chain via the action of PmCS. The chimeric molecule was then treated with HA lyase, digesting the HA chain, leaving the chondroitin chain component intact.
We did note that the entire bulk chondroitin sulfate polymer starting material was not incorporated into chimeric molecules even after repeated addition of synthase and UDP-sugars; this observation suggests that certain nonreducing termini (espe-cially the 4-sulfated chondroitin units) cannot be efficiently extended by native sequence PmHAS or PmCS enzymes. At the start of a reaction utilizing a poorly functioning noncognate acceptor, the Pasteurella synthase slowly extends the polymer, but once the catalyst adds on a short cognate sugar extension to the nonreducing terminus, the nascent molecule is transformed rapidly into an excellent acceptor. At longer times (e.g. approximately Ͼ2 h), the differences because of the initial lag period are obscured, and thus the differences among the various chondroitin sulfate preparations are less apparent.
The only other reported methods for chemoenzymatically constructing chimeric GAGs involved the use of testicular hyaluronidase to (a) transglycosylate GAG chains (28,29) or (b) couple oxazoline monomers (30). Testicular hyaluronidase catalyzes the cleavage of HA, chondroitin, or chondroitin sulfate using a water hydroxyl with an optimum at pH 5. However, the hyaluronidase can also use a polymer hydroxyl group thus adding saccharide units instead of cleaving in a reaction called transglycosylation with an optimum at pH 7. Unfortunately, the transglycosylation method is difficult to control and has low yields; a mixture of products (i.e. a series of structurally similar chimeric GAGs with ϳ6 -22 saccharide units) result, and the degrading enzyme will actually cleave the products. In the latter elegant method, oxazoline sugar analogs (e.g. employing mixtures of HA, chondroitin, or chondroitin sulfate disaccharides, etc.) mimicking the transition state are coupled by the hyaluronidase. However, these analogs are unstable in water resulting in a potential overabundance of "dead-end" acceptors (the oxazoline analog degrades to an ordinary disaccharide) compared with the activated donor that drives down the size distribution of the final products (ϳ10 -20 kDa or ϳ50 -100 sugars reported). In addition, the product degradation by hyaluronidase problem mentioned above in method a also exists. Also, the exact placement of desired sugars within a given target structure (especially for small chains) is difficult or impossible for the oxazoline process. On the other hand, the HA and chondroitin synthases and their mutants may be utilized in a stepwise fashion to make defined GAG oligosaccharides (14).
Proteoglycan-like molecules with two distinct high molecular weight GAG components, but missing the protein bridge (e.g. core and link proteins), can be assembled by our method. These molecules may potentially serve the field of tissue engineering as scaffolds to assemble various cell types into tissues or organs. The lack of any polypeptide in these artificial chimeric molecules removes the issues of protease susceptibility and concerns of both immunogenicity and allergenicity (e.g. when an animal-derived product is used in humans). Furthermore, the threat of adventitious agents (e.g. prions, virus) from animal sources may be diminished or eliminated.
On a smaller scale, novel oligosaccharides with HA-like and/or chondroitin-like structures may also be constructed using the Pasteurella synthases. 3 We are exploring if these molecules bind with different selectivity or affinity to hyaladherins, HA-binding proteins, or receptors. In addition to mapping out the oligosaccharide binding requirements of hyaladherins, we 3 B. S. Tracy, A. E. Sismey, and P. L. DeAngelis, unpublished observations. may be able to generate compounds that will selectively inhibit the binding of HA to one particular hyaladherin species without perturbing other species. Such sugar molecules may have future utility as selective therapeutics with minimal side effects for diseases such as cancer, autoimmune disease, inflammation, and infection.